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超高效液相色谱-串联质谱法同时检测地龙药材中4种黄曲霉毒素含量及阳性结果确证

分析测试经验介绍 (104 ~ 109)超高效液相色谱-串联质谱法同时检测地龙药材中4种黄曲霉毒素含量及阳性结果确证郭　晶1，张　进1，李文君1，李正刚2（1. 通化市食品药品检验所，吉林通化　134000；2. 四平市食品药品检验所，吉林四平　136000）摘要：建立超高效液相色谱-串联质谱法同时测定地龙药材中黄曲霉毒素B 1、B 2、G 1、G 2的含量，并对阳性结果进行确证. 样品经70%甲醇提取、免疫亲和柱净化、超高效液相色谱柱分离、三重四极杆质谱检测，并采用离子峰度比进行阳性结果确证. 结果表明，4种黄曲霉毒素的线性关系良好（r >0.999 5），回收率在91.2%~95.6%范围内.黄曲霉毒素B 1、B 2、G 1、G 2的检出限分别为0.10、0.038、0.11、0.038 µg/kg. 方法专属性好，灵敏度高，准确性好，可以进行最终阳性检出的判定. 15批地龙药材中6批黄曲霉毒素确证阳性检出, 证明地龙药材较易被黄曲霉毒素污染.关键词：地龙；超高效液相色谱-串联质谱；黄曲霉毒素；阳性结果确证中图分类号：O657. 63 文献标志码：B 文章编号：1006-3757（2024）02-0104-06DOI ：10.16495/j.1006-3757.2024.02.005Simultaneous Determination of Four Aflatoxins in Pheretima by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry andConfirmation of Positive ResultsGUO Jing 1， ZHANG Jin 1， LI Wenjun 1， LI Zhenggang2（1. Tonghua Institute for Food and Drug Control , Tonghua 134000, Jilin China ；2. Siping Institute for Food andDrug Control , Siping 136000, Jilin China ）Abstract ：A method for simultaneous determination of aflatoxins B 1, B 2, G 1, and G 2 in pheretima was been established by ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), and the positive results was confirmed. The samples were extracted by 70% methanol, purified by a immunoaffinity columns, separated by UPLC-MS/MS, detected by triple quadrupole mass spectrometry, and confirmed by ion peak ratio. The results showed that the linear relationship of the four aflatoxins were good (r >0.999 5), and the recoveries ranged from 91.2% to 95.6%. The limits of detection for aflatoxin B 1, B 2, G 1, and G 2 were 0.10, 0.038, 0.11, 0.038 µg/kg, respectively. The method has a good specificity, high sensitivity, good accuracy, and can be used to determine the final positive detection. Aflatoxin were positively detected in 6 out of 15 batches of pheretima , which proved that the Pheretima were more susceptible to contamination by aflatoxins.Key words ：Pheretima ；UPLC-MS/MS ；aflatoxin ；confirmation of positive results收稿日期：2024−01−10；　修订日期：2024−03−05.基金项目：吉林省中药材标准及中药饮片炮制规范（项目编号：JLPZGF-2020-052）作者简介：郭晶（1979−），女，副主任药师，研究方向为药品质量标准通信作者：李正刚（1982−），男，硕士，医药工程师，研究方向为中药质量标准，E-mail ：*****************.第 30 卷第 2 期分析测试技术与仪器Volume 30 Number 22024年3月ANALYSIS AND TESTING TECHNOLOGY AND INSTRUMENTS Mar. 2024黄曲霉毒素（aflatoxins, AF）是一种毒性极强的剧毒物质，被各个国家的癌症研究机构列为Ⅰ类致癌物. 黄曲霉毒素主要是由黄曲霉、寄生曲霉产生的次生代谢产物，是一组化学结构类似的化合物[1-2]，主要包括AFB1、AFB2、AFG1、AFG2，多存在于土壤、动植物、各种坚果中[3]，在湿热环境下出现黄曲霉毒素污染的概率最高. 黄曲霉毒素的危害性在于对人及动物肝脏组织的慢性毒性作用，长期积累可导致肝癌甚至死亡[4-5]. 各国药典均对黄曲霉毒素在中药材或中草药中的限量作了规定，如《欧洲药典》规定AFB1、AFB2、AFG1、AFG2总量在中草药中的限量不得高于4 µg/kg，《美国药典》规定AFB1、AFB2、AFG1、AFG2总量在中草药中的限量不得高于20µg/kg，《中华人民共和国药典》规定其总量在中药材中的限量不得高于10 µg/kg[6].地龙药材标准收载在《中华人民共和国药典》2020版一部[7]. 采收时需要及时剖开腹部，除去内脏和泥沙. 由于地龙生长在土壤环境中，故其被AF污染的风险较大. 目前AF测定的方法主要有液相色谱-柱后碘衍生化法或光化学衍生化法-荧光检测器以及酶联免疫法，但以上方法均存在假阳性的误判可能性，当超出标准限值时均需要采用质谱检测器进行阳性确证[8]. 本研究采用超高效液相色谱-串联质谱法（UPLC-MS/MS）同时测定不同产地多批次地龙药材中AFB1、AFB2、AFG1、AFG2的含量，并对阳性结果进行确证，从而准确的判定检出情况，为地龙药材中AF污染情况提供参考.1　试验部分1.1　仪器和试剂BT125D型电子天平（北京赛多利斯仪器天平有限公司），TDL-5-A型离心机（上海安亭科学仪器厂），AB SCIEX QTRAP® 5500型超高效液相色谱-三重四极杆质谱（美国AB SCIEX公司）. 超高压液相色谱柱（岛津科技有限公司），免疫亲和柱（青岛普瑞邦生物工程有限公司，规格：3 mL，批号：2J1J24-4）.AF混合对照品溶液：AFB1、AFB2、AFG1、AFG2标示质量浓度分别为1.04、0.38、1.08、0.38µg/mL，批号：610001-202006，来源于中国食品药品检定研究院. 氯化钠和醋酸铵（国药集团化学试剂有限公司，分析纯），甲醇和乙腈（Flsher Scientific，色谱纯），纯化水为自制.本次收集到不同产地和批次的地龙药材共15批，基本信息如表1所列.表 1 地龙药材样品基本信息Table 1　Basic informations of Pheretima samples药材编号样品名称产地DL1广地龙海南文昌DL2广地龙广东肇庆DL3广地龙广东韶关DL4广地龙广东韶关DL5广地龙广西玉林DL6广地龙广西北海DL7沪地龙湖北襄阳DL8沪地龙河南濮阳DL9沪地龙河南安阳DL10沪地龙浙江嘉兴DL11沪地龙河南焦作DL12沪地龙浙江金华DL13沪地龙江西赣州DL14沪地龙江西九江DL15沪地龙江西宜春1.2　仪器工作条件1.2.1　色谱条件色谱柱：Shim-pack XR-ODS，3.0 mm×75 mm （1.8 µm）；以10 mmol/L醋酸铵溶液为流动相A，甲醇为流动相B；柱温：25 ℃；流速：0.3 mL/min. 按表2中的程序进行梯度洗脱.表 2 梯度洗脱程序Table 2　Gradient elution procedure时间/min流动相A/%流动相B/%0~4.565~1535~854.5~615~085~1006~6.50~65100~356.5~1065351.2.2　质谱条件离子源：电喷雾离子源（ESI源）；监测模式：正离子扫描下的多反应监测模式（MRM）；喷雾压强：0.21 MPa；干燥气流速：8 L/min；干燥气温度：550 ℃；毛细管电压：5 500 V；三重四极杆离子对及相关电压参数设定如表3所列.第 2 期郭晶，等：超高效液相色谱-串联质谱法同时检测地龙药材中4种黄曲霉毒素含量及阳性结果确证1051.3　标准曲线绘制分别精密量取AF混合对照品溶液100、50、25µL于10 mL容量瓶中，用甲醇稀释至刻度，摇匀，即得标准曲线溶液1、2、3. 分别精密量取1 mL标准曲线溶液2、0.1 mL标准曲线溶液1于10 mL容量瓶中，用甲醇稀释至刻度，摇匀，即得标准曲线溶液4、5. 精密量取1 mL标准曲线溶液4于10 mL 容量瓶中，用甲醇稀释至刻度，摇匀，即得标准曲线溶液6.1.4　供试品溶液制备取供试品粉末约15 g（过四号筛网），精密称定，置于均质瓶中，加入氯化钠3 g、70%甲醇溶液75 mL，12 000 r/min高速搅拌2 min，4 500 r/min离心5 min，取上清液15.0 mL，置于50 mL量瓶中，用水稀释至刻度，摇匀，4 500 r/min离心10 min，取续滤液20.0 mL. 通过免疫亲合柱，流速3 mL/min，用20 mL水洗脱，洗脱液弃去，使空气进入免疫亲合柱，将水挤出，再用适量甲醇洗脱，收集洗脱液，置于2 mL量瓶中，加甲醇稀释至刻度，摇匀，用微孔滤膜（0.22 µm）滤过，取续滤液，即得.2　结果与讨论2.1　免疫亲和柱技术和质谱测定法的优势免疫亲和柱技术是利用抗原、抗体之间高度特异性结合对目标物进行净化富集的一种实验技术，预置在柱体内的特异性抗体选择性结合样本中的待测物，杂质不被结合流出柱外，柱上结合的目标物再被洗脱液洗脱，从而实现目标物的高效净化和浓缩[9].质谱法是使待测化合物产生气态离子，再按质荷比（m/z）将离子分离、检测的分析方法，检测限可达10−15~10−12 mol数量级. 相比较液相色谱法，测试样品需要的量较少，其检测灵敏度也大幅度提高，且其较好的专属性可以作为液相色谱检出情况的进一步确证，大大降低误判阳性检出的情况[10-13]. 2.2　线性范围和检出限分别精密吸取1.3项下系列混合对照品溶液5µL，注入液相色谱仪，测定峰面积，以进样质量浓度（ng/mL）为横坐标，峰面积为纵坐标，绘制标准曲线.结果如表4所列，4种黄曲霉毒素的线性关系良好（r>0.999 5）.取不含AF的样品15 g，加入0.15 mL标准曲线溶液1，余下操作同1.4项下供试品溶液制备，1.2项下仪器工作条件测定，以信噪比（S/N）为3作为4种黄曲霉毒素的检出限（limit of detection，LOD），结果如表4所列，AFB1、AFB2、AFG1、AFG2的LOD分别为0.10、0.038、0.11、0.038 µg/kg，表明方法灵敏度较高.表 4 回归方程、线性范围和检出限Table 4　Regression equations, linear ranges and limits of detection组分名称回归方程r线性范围/（ng/mL）LOD/（µg/kg）S/N AFB1y=33 600x –1 3600.999 90.104～10.4000.10 4.5AFB2y=30 900x – 4 8800.999 80.038～3.8000.038 3.1AFG1y=26 150x – 6 3400.999 80.108～10.8000.11 4.2AFG2y=21 200x + 2 9200.999 80.038～3.8000.038 3.32.3　精密度考察取5 µL 1.3项下标准曲线溶液1，在1.2项下仪器条件及测定方法下分别连续进样6次，测定各组分的色谱峰面积. 测试结果：AFB1、AFB2、AFG1、表 3 质谱条件Table 3　Conditions of mass spectrometer序号组分名称母离子/（m/z）子离子/（m/z）碎裂电压/V碰撞能量/eV1AFB1313.1285.1*16034241.0207512AFB2315.1287.1*18937259.1200393AFG1329.1243.1*16338311.1180314AFG2331.1313.1*18535245.118842注：*定量离子对106分析测试技术与仪器第 30 卷AFG2色谱峰面积相对标准偏差（RSD)分别为1.3%、2.2%、1.8%和2.2%，表明仪器的精密度良好.2.4　稳定性试验取1.3项下标准曲线溶液1，分别于0、4、８、16、18、24 h 按1.2项下仪器条件进样5 µL，记录所测各组分峰面积. 测试结果：AFB1、AFB2、AFG1、AFG2色谱峰面积RSD分别为2.6%、2.2%、2.5%和2.3%，结果表明在24 h内测定结果稳定.2.5　重复性和回收率试验由于15批次地龙药材中仅部分检出AFB1、AFB2，故在加标回收试验中进行方法的重复性考察.称取不含AF的地龙药材（编号：DL1）粉末约15 g，各6份，分别精密加入AF混合对照品溶液75 µL，其余操作同2.1.2项下供试品溶液制备，按2.2项下条件分别测定各组分的色谱峰面积，结果如表5所列. 由表5可见，AFB1、AFB2、AFG1、AFG2的平均回收率分别为95.6%、93.4%、93.2%、91.2%，加标测定值的RSD分别为1.9%、2.0%、1.7%和2.6%，表明该方法的回收率满足要求，方法的重复性良好.2.6　样品测定取15批次地龙药材，按照1.4项下方法制备供试品溶液，1.2项下仪器条件进行测定，结果如表6所列，结果表明在6批地龙样品中检测出AFB1和AFB2，质谱总离子流图如图1所示.表 6 样品测定结果Table 6　Determination results of samples/(µg/kg)样品编号AFB1质量分数AFB2质量分数AFG1质量分数AFG2质量分数总质量分数DL1/////DL2/////DL3/////DL4 6.80.53//7.33 DL5/////DL6/////DL77.80.35//8.15 DL8 6.50.31// 6.81 DL9 4.30.24// 4.54 DL10/////DL11/////DL12/////DL13 3.10.43// 3.53 DL14 2.50.31// 2.81 DL15/////注：“/”：未检出2.7　阳性结果确证中药组成成分相较于西药组成成分复杂多样，三重四极杆质谱是以母离子及子离子组成的离子对进行检测，由于样品基质成分的复杂性，单个离子对出现干扰的情况在试验过程中偶有发生. 然而一个化合物在质谱检测器的离子源中被固定碰撞电压击碎产生的多个碎片离子之间具有相对固定的比例，故采用离子对峰面积的比值进行比较更具有专属性，UPLC-MS/MS法通常采用离子峰度比对结果进一步确证阳性检出，即：如果样品检出色谱峰的保留时间与对照品一致，并且在扣除背景后的表 5 方法的重复性和回收率试验结果Table 5　Experimental results of repeatability andrecovery of method添加质量分数加入质量/ng实测质量/ng回收率/%平均回收率/%RSD/%AFB1 (5 µg/kg)78.0074.2195.1495.6 1.9 78.0073.1193.7378.0075.5396.8378.0076.5298.1078.0072.9993.5878.0075.2396.45AFB2 (2 µg/kg)28.5026.1291.6593.4 2.0 28.5025.2390.7728.5026.7893.9628.5027.0194.7728.5027.3295.8628.5026.5593.16AFG1 (5 µg/kg)81.0077.4495.6093.2 1.7 81.0075.2692.9181.0075.3693.0481.0074.8292.3781.0076.3594.2681.0073.7891.09AFG2 (2 µg/kg)28.5025.1591.0591.2 2.6 28.5026.2292.0028.5025.4789.3728.5025.1188.1128.5027.0194.7728.5026.2892.21第 2 期郭晶，等：超高效液相色谱-串联质谱法同时检测地龙药材中4种黄曲霉毒素含量及阳性结果确证107质谱图中，所选择的监测离子对均出现，而且所选择的监测离子对峰面积比与对照品的监测离子对峰面积比一致，则可判定样品中存在该真菌毒素.经计算本试验检出的6批阳性样品的离子峰度比AFB 1分别为75.3%、75.2%、75.1%、75.3%、75.2%、75.1%，相对平均偏差均小于0.2%. AFB 2分别为85.0%、85.1%、85.1%、85.2%、85.3%、85.0%，相对平均偏差均小于0.2%. 均在允许偏差±20%内，确证样品阳性检出.3　结论由多批次样品检验情况可以初步得出，地龙药材中黄曲霉毒素的污染主要为黄曲霉毒素B 1和B 2.地龙药材在产地初加工过程中需去除泥沙，而泥沙为黄曲霉毒素污染的重要来源，如果泥沙去除不净、保存不当、遇湿热环境，则黄曲霉毒素含量容易超标[14-17]. 因此地龙药材的黄曲霉毒素监测检验非常必要[18-21]. 试验结果表明，UPLC-MS/MS 法操作简便、专属性好、灵敏度高、重复性好，能够对地龙药材的阳性结果作出准确判定，故将在中药材的黄曲霉毒素污染检测中发挥重要作用.参考文献：刘丽娜, 李海亮, 李耀磊, 等. 中药真菌毒素质量控制概况、限量标准制定及有关问题的思考[J ]. 中草药，2023，54（19）：6197-6207. [LIU Lina, LI Hailiang, LI Yaolei, et al. Overview on quality control of mycotox-ins in traditional Chinese medicine, limit requirements and discussion on related issues [J ]. Chinese Tradition-al and Herbal Drugs ，2023，54 （19）：6197-6207.]［ 1 ］沈立, 汤燕, 陈铁柱, 等. 固相萃取液质联用测定川产道地药材黄精、麦冬中10种真菌毒素[J ]. 药物分析杂志，2023，43（8）：1369-1380. [SHEN Li, TANG［ 2 ］Yan, CHEN Tiezhu, et al. Determination of 10 my-cotoxins in Polygonati Rhizoma and Ophiopogonis Radix as a genuine regional drug of Sichuan by solid-phase extraction coupled with LC-MS/MS [J ]. Chinese Journal of Pharmaceutical Analysis ，2023，43 （8）：1369-1380.]徐晓艳, 王宇, 王梦瑶, 等. 中药材中真菌毒素的检测与脱毒研究进展[J ]. 中草药，2024，55（2）：657-669.[XU Xiaoyan, WANG Yu, WANG Mengyao, et al.Research progress on detection and detoxification of mycotoxins in Chinese medicinal materials [J ].Chinese Traditional and Herbal Drugs ，2024，55 （2）：657-669.]［ 3 ］李正刚, 程焱, 王丹彧, 等. 地龙药材中黄曲霉毒素B 1、B 2、G 1、G 2的含量检测[J ]. 化学工程师，2023，37（8）：34-37. [LI Zhenggang, CHENG Yan, WANG Danyu, et al. Detection of aflatoxins B 1、B 2、G 1、G 2 in Pheretima materials [J ]. Chemical Engineer ，2023，37（8）：34-37.]［ 4 ］莫紫梅. 黄曲霉毒素B 1降解产物及其安全性评价研究进展[J/OL ]. 中国油脂, 2023:1-12[2024-02-29].https:///10.19902/ki.zgyz.1003-7969.230329. [MO Zimei. Research progress on degrada-tion products of aflatoxin B 1 and its safety evaluation [J/OL ]. China Oils and Fats, 2023:1-12[2024-02-29]. https:///10.19902/ki.zgyz.1003-7969.230329.]［ 5 ］孙艳杰, 李正刚, 赵磊, 等. 制貂肾中黄曲霉毒素的测定方法研究及暴露风险评估[J ]. 中国药物评价，2023，40（3）：249-252. [SUN Yanjie, LI Zhenggang,ZHAO Lei, et al. Determination method of aflatoxin in processed mustela and risk assessment [J ]. Chinese Journal of Drug Evaluation ，2023，40 （3）：249-252.]［ 6 ］国家药典委员会. 中华人民共和国药典(一部)[S ].北京: 中国医药科技出版社, 2020: 127.［ 7 ］许晓辉, 李晨曦, 吴福祥, 等. QuEChERS-分散固相萃取-液质联用法快速测定地龙中黄曲霉毒素[J ].分析测试技术与仪器，2021，27（1）：18-23. [XU［ 8 ］1×1042×1043×104(a)(b)4×1045×1046×1047×104t /minI n t e n s i t y /c p sI n t e n s i t y /c p st /min1×1042×1043×1044×1045×1046×104AFB 2AFB 1AFG 2AFB 2AFB 1AFG 1图1　样品测定图谱（a ）对照品质谱总离子流图，（b ）样品质谱总离子流图Fig. 1　Total ion flow diagrams of samples108分析测试技术与仪器第 30 卷Xiaohui, LI Chenxi, WU Fuxiang, et al. QuEChERS-dispersed solid phase extraction-ultra performance li-quid chromatography-tandem mass spectrometry for rapid determination of aflatoxin in pheretima [J ]. Ana-lysis and Testing Technology and Instruments ，2021，27 （1）：18-23.]马彧, 李正刚, 程焱, 等. 超声萃取-免疫亲和柱净化-柱后光化学衍生高效液相色谱法同时测定蜂房药材中4种黄曲霉毒素[J ]. 化学分析计量，2023，32（2）：20-23, 56. [MA Yu, LI Zhenggang, CHENG Yan, et al. Simultaneous determination of four aflatox-ins in Nidus Vespae by ultrasonic extraction-immun-oaffinity column purification-post column photochem-ical derivatization HPLC [J ]. Chemical Analysis and Meterage ，2023，32 （2）：20-23, 56.]［ 9 ］龚敏, 陈明亮, 金鹏, 等. QuEChERS 结合UPLC-MS/MS 测定水产品中4种黄曲霉毒素及其裂解规律研究[J ]. 热带农业科学，2023，43（10）：35-40.[GONG Min, CHEN Mingliang, JIN Peng, et al. De-termination of four aflatoxins in aquatic product by QuEChERS combine UPLC-MS/MS method and study on fragmentation pathway [J ]. Chinese Journal of Tropical Agriculture ，2023，43 （10）：35-40.]［ 10 ］袁正宁, 李梁子涵, 王艳敏, 等. 黄曲霉毒素对肝脏和脾脏的毒理作用研究[J ]. 中国畜禽种业，2023，19（4）：91-95. [YUAN Zhengning, LI Liangzihan,WANG Yanmin, et al. Toxicological effects of aflatoxin on liver and spleen [J ]. The Chinese Live-stock and Poultry Breeding ，2023，19 （4）：91-95.]［ 11 ］何洁, 余婷婷, 梁松, 等. 高通量高效快速净化方法结合UPLC-MS/MS 测定粮食中黄曲霉毒素[J/OL ]. 中国测试, 2023:1-12 [2024-02-29]. https:///kcms/detail/51.1714.TB.20230421.1514.008.html.[HE Jie, YU Tingting, LIANG Song, et al. Determina-tion of aflatoxins in grains using the high-throughput high efficient flow-through clean-up method and ultra-high performance liquid chromatography tandem mass spectrometry [J/OL ]. China Measurement & Test,2023:1-12 [2024-02-29]. https:///kcms/de-tail/51.1714.TB.20230421.1514.008.html.]［ 12 ］李正刚, 谢秋红, 邵大志, 等. 高效液相色谱-荧光检测法同时测定人参归脾丸中4种黄曲霉毒素[J ]. 中国卫生工程学, 2022, 21(5):714-716. [LI Zhenggang,XIE Qiuhong, SHAO Dazhi, et al. Simultaneous de-termination of content of four Aflatoxins Ginseng spleen-invigorating bolus by HPLC-FLD [J ]. Chinese Journal of Public Health Engineering, 2022, 21(5):714-716.]［ 13 ］陈莉. 超高效液相色谱串联三重四极杆质谱法同时测定舒眠胶囊(片)中4种黄曲霉毒素含量[J ]. 中国药业，2022，31（22）：77-80. [CHEN Li. Simultaneous［ 14 ］determination of four aflatoxins in Shumian capsules(tablets) by UPLC-QQQ/MS [J ]. China Phar-maceuticals ，2022，31 （22）：77-80.]夏仕青, 李晓慧, 吴桃丽. 超高效液相色谱-串联质谱法测定辣椒粉中4种黄曲霉毒素的含量[J ]. 微量元素与健康研究，2023，40（1）：57-60. [XIA Shiqing, LI Xiaohui, WU Taoli. Determination of four aflatoxins in Chili Powder by UPLC-MS/MS [J ]. Studies of Trace Elements and Health ，2023，40 （1）：57-60.]［ 15 ］淦露. 黄曲霉毒素检测技术研究进展[J ]. 现代食品，2022，28（15）：114-117. [GAN Lu. Research progress of aflatoxin detection technology [J ]. Modern Food ，2022，28 （15）：114-117.]［ 16 ］杨昊, 公丕学, 王骏, 等. 基于石墨烯净化的超高效液相色谱串联质谱快速测定乳制品中黄曲霉毒素[J ].中国食品卫生杂志，2023，35（3）：396-402. [YANG Hao, GONG Pixue, WANG Jun, et al. Fast determina-tion of aflatoxin in dairy products by ultra-high per-formance liquid chromatography-tandem mass spec-trometry based on purification of graphene [J ].Chinese Journal of Food Hygiene ，2023，35 （3）：396-402.]［ 17 ］赵飞. QuEChERS 结合超高效液相色谱-三重四极杆质谱法测定坚果中黄曲霉毒素[J ]. 当代化工，2022，51（3）：752-756. [ZHAO Fei. Determination of aflatoxins in nuts by QuEChERS coupled with ultra-li-quid chromatography-mass spectrometry [J ]. Contem-porary Chemical Industry ，2022，51 （3）：752-756.]［ 18 ］谭丽盈. 超高效液相色谱-串联质谱法同时测定醋延胡索药材中4种黄曲霉毒素[J ]. 化学分析计量，2021，30（10）：27-32. [TAN Liying. Simultaneous de-termination of four kinds of aflatoxins in vinegar cory-dalis by UPLC-MS/MS [J ]. Chemical Analysis and Meterage ，2021，30 （10）：27-32.]［ 19 ］周颖, 祝清岚, 马临科, 等. 免疫亲和柱净化-柱后光化学衍生-高效液相色谱法检测蜚蠊中的黄曲霉毒素[J ]. 中国药物评价，2020，37（5）：358-361.[ZHOU Ying, ZHU Qinglan, MA Linke, et al. De-termination of aflatoxins in periplaneta Americana by immunoaffinity column and high performance liquid chromatography coupled with post-column photo-chemical derivatization [J ]. Chinese Journal of Drug Evaluation ，2020，37 （5）：358-361.]［ 20 ］刘宁, 王萌萌, 金红宇, 等. 高效液相色谱-柱后光化学衍生法测定参苓白术散中黄曲霉毒素G 2、G 1、B 2、B 1[J ]. 中国药事，2012，26（5）：442-445. [LIU Ning,WANG Mengmeng, JIN Hongyu, et al. Determination of aflatoxin G 2, G 1, B 2, B 1 in Shenling Baizhu Powder by HPLC with post column photochemical derivatiza-tion [J ]. Chinese Pharmaceutical Affairs ，2012，26 （5）：442-445.]［ 21 ］第 2 期郭晶，等：超高效液相色谱-串联质谱法同时检测地龙药材中4种黄曲霉毒素含量及阳性结果确证109。

磁力架说明书_Magnetic Stand Manual_MCE

1包装清单产品概述MCE 磁力架是磁珠产品专用配套设备，支持 MCE 全线磁珠类产品，内含强磁磁芯，可实现快速高效的分离。

MCE 磁力架 (200 μL / 2 mL / 15 mL ) 采用独特的三明治槽设计，磁条可抽出，可容纳 200 μL PCR 管，1.5 mL EP 管，2 mL EP 管，15 mL 离心管。

本产品适用于抗体纯化、免疫沉淀 (IP )、免疫共沉淀 (Co-IP )、细胞分选和核酸分离等实验。

2操作说明31. 将装有磁珠悬液的 EP 管/离心管置于磁力架对应的样品孔中，静置数分钟后磁珠被吸附聚集于管壁，溶液恢复澄清。

2. 用移液器或吸管从管底将溶液吸出，或小心倾倒出液体。

3. 抽去磁力条，加入复溶液体，轻缓震荡即可混合均匀，进行下一步操作。

注：磁性分离的时间与磁珠粒径有关，磁珠粒径越小，磁性分离时间越长。

此外，溶液的黏稠程度以及溶液的成分也会对磁性分离时间产生影响。

5注意事项1. 根据实验参数和样品体积不同，可调整磁芯位置或选用不同样式的磁力架。

2. 为减少操作过程中磁珠的损失，请将样品管底端插入磁力架底部的凹槽内。

当磁珠吸附在管壁上后，缓慢倾去上清，或用移液枪吸尽。

3. 由于磁力架有强大的磁场，请远离手机、电脑、手表、起博器、磁铁等易被磁力干扰的物体，尤其是刀具，以免对操作人员造成伤害。

4. 如需同时使用多个磁力架 (≥2 个) ，应分开放置，避免磁场之间产生干扰。

不要把多个磁棒放在一起，以防止夹伤。

5. 请勿与强酸、强碱等腐蚀性溶剂直接接触。

6. 请勿拆卸磁块。

7. 为保护外壳，请勿长时间暴露在阳光和紫外线下。

8. 为保持磁力架磁性，请勿置于高温和强外界磁场环境中。

9. 使用后请及时清洁，妥善放置在干燥环境中。

Magnetic StandMedChemExpress MedChemExpress 400-820-3792 电话: ************ 传真: ************Email: t *********************MCE Hotline: 400-820-3792Contents HY-K0200Magnetic Stand 200 μL-2 mL-15 mL。

BFH772_DataSheet_MedChemExpress

Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:BFH772 is a potent oral VEGFR2 inhibitor, which is highly effective at targeting VEGFR2 kinase with an IC 50 value of 3 nM.IC50 & Target: IC50: 2.7±0.9 nM (hVEGFR2), 1.5±0.53 μM (mVEGFR2), 1.7±0.36 μM (hVEGFR1), 1.1±0.29 μM (hVEGFR3)[1]In Vitro: BFH772 is highly selective; apart from inhibiting VEGFR2 at 3 nM IC 50, it also targets B–RAF, RET, and TIE–2, albeit with atleast 40–fold lower potency. BFH772 is inactive (IC 50>10 μM; >2 μM for cKIT) against all other tyrosine specific– andserine/threonine–specific protein kinases tested. BFH772 inhibits VEGFR2 with IC 50 of 4.6±0.6 nM in CHO cells. BFH772 inhibits VEGFR2 with IC 50 of 3 nM in HUVEC cells. BFH772 inhibits the ligand induced autophosphorylation of RET, PDGFR, and KIT kinases,with IC 50 values ranging between 30 and 160 nM. BFH772 is selective (IC 50 values >0.5 μM) against the kinases of EGFR, ERBB2,INS–R, and IGF–1R and against the cytoplasmic BCR–ABL kinase. IC 50 of BFH772 (<0.01 nM, n=2) demonstrates that they abrogated VEGF induced proliferation at remarkably low nM concentrations [1].In Vivo: BFH772 at 3 mg/kg orally dosed once per day potently inhibits melanoma growth (by 54–90% for primary tumor and71–96% for metastasis growth) as depicted by treatment to control ratios. Dose–response curves of BFH772 at 0.3, 1, and 3 mg/kg demonstrate that even at the lowest concentrations, this naphthalene–1–carboxamide inhibits VEGF induced tissue weight and TIE–2 levels but only reaches statistical significance at 1 mg/kg and above [1].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[1]In vitro kinase assay is based on a filter binding assay, using the recombinant GST–fused kinase domainsexpressed in baculovirus and purified over glutathione–sepharose, γ–[33P]ATP as the phosphate donor, and poly(Glu:Tyr 4:1) peptide as the acceptor. Each GST–fused kinase is incubated under optimized buffer conditions [20 mM Tris–HCl buffer (pH 7.5), 1–3 mM MnCl 2, 3–10 mM MgCl 2, 3–8 μg/mL poly(Glu:Tyr 4:1), 0.25 mg/mL polyethylene glycol 20000, 8 μM ATP, 10 μM sodium vanadate, 1mM DTT] and 0.2 μCi γ–33P ATP in a total volume of 30 μL in the presence or absence of a test substance for 10 min at ambient temperature. The reaction is stopped by adding 10 mL of 250 mM EDTA. Using a 384–well filter system, half the volume istransferred onto an Immobilon–polyvinylidene difluoride membrane. The membrane is then washed extensively and dried, and scintillation counting is performed. IC 50s for compounds are calculated by linear regression analysis of the percentage inhibition [1].Cell Assay: BFH772 is dissolved in DMSO (10 mM) and stored, and then diluted with appropriate medium before use [1]. [1]DifferentBa/F3 cell lines rendered IL–3 independent by transduction with various constitutively active tyrosine kinases are grown in RPMI 1640 medium containing 10% fetal calf serum. For maintenance of parental Ba/F3 cells, the medium is additionally supplemented with 10 ng/mL interleukin–3 (IL–3). For proliferation assays, Ba/F3 cells are seeded on 96–well plates in triplicates at 10000 cells per well and incubated with various concentrations of compounds for 72 h followed by quantification of viable cells using a resazurin sodium salt dye reduction readout (commercially known as Alamar Blue assay). IC 50s are determined with the XLFit Excel Add–In using a four–parameter dose response model [1].Animal Administration: BFH772 is prepared in PEG200 100% (Mice)[1].Product Name:BFH772Cat. No.:HY-100419CAS No.:890128-81-1Molecular Formula:C 23H 16F 3N 3O 3Molecular Weight:439.39Target:VEGFR Pathway:Protein Tyrosine Kinase/RTK Solubility:DMSO: 7.75 mg/mLBFH772 is dissolved in N–methyl pyrrolidone/polyethylene glycol200 (30:70, v/v) (Rat)[1].[1]Mice[1]Female FVB mice weighing between 18 and 20 g are housed in groups of six. Porous chambers containing VEGF (2 μg/mL) in 0.5 mL of 0.8% w/v agar (containing heparin, 20 U/mL) are implanted subcutaneously in the flank of the mice (n=6 per group). VEGF induces the growth of vascularized tissue around the chamber. This response is dose–dependent and can be quantified by measuring the weight and TIE–2 levels of the tissue. Mice are treated either orally once daily with compounds or vehicle (PEG200 100%, 5 mL/kg) starting4–6 h before implantation of the chambers and continuing for 4 days. The animals are sacrificed for measurement of the vascularized tissues 24 h after the last dose. Tissue weight is taken and then a lysate prepared for TIE–2 ELISA analysis .Rat[1]Catheters are implanted into the femoral artery and vein of na?ve female rats strain OFA for BFH772, and BAW2881, or in the jugular vein and femoral artery in female Sprague–Dawley rats for compounds 4, 9, and 10. Animals are allowed to recover for 96 h and are housed in single cages with free access to food and water throughout the experiment. Female OFA rats received 2.5 mg/kg ofBAW2881 dissolved in ethanol/dimethylisosorbide/polyethylene glycol400/D5W (10/15/35/40 v/v) or 1 mg/kg of BFH772 dissolved in N–methyl pyrrolidone/polyethylene glycol200 (30:70, v/v) via injection into the femoral vein. D5W is glucose 5%/water (v/v). Oral administration: BAW2881 and BFH772 are formulated as a micronized suspension (dissolved/suspended in 0.5% carboxymethyl cellulose in distilled water) and administered by gavage to female OFA rats to deliver a dose of 25 mg/kg for BAW2881 or 3 mg/kg BFH772 (n=4 rats per group). For compounds 4, 9, and 10, female Sprague–Dawley rats at 8 weeks of age received an intraveno References:[1]. Bold G, et al. A Novel Potent Oral Series of VEGFR2 Inhibitors Abrogate Tumor Growth by Inhibiting Angiogenesis. J Med Chem. 2016 Jan 14;59(1):132–46.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

T0070907_LCMS_04465_MedChemExpress

=====================================================================Acq. Operator : Li Shan(LCMS-02) Seq. Line : 50Acq. Instrument : HY-LCMS-02 Location : P1-C-01Injection Date : 5/15/2015 12:20:58 PM Inj : 1Inj Volume : 3.000 µlAcq. Method : D:\AGLIENT 1260\DATA\20150515\20150515 2015-05-15 08-47-06\100-1000MS+3MIN-1.5_(0.02%FA).MLast changed : 5/15/2015 8:47:06 AM by Li Shan(LCMS-02)Analysis Method : D:\AGLIENT 1260\METHOD\BL30-90A,210NM(RP-HPLC).MLast changed : 5/15/2015 3:12:48 PM by Li Shan(LCMS-02)(modified after loading)M ethod Info : HPLC Catalog No : HY-13202 Batch#04465 A-RP-132Additional Info : Peak(s) manually integratedmin0.51 1.52 2.53mAU20040060080010001200DAD1 C, Sig=254,4 Ref=off (D:\AGLIENT...0\DATA\20150515\20150515 2015-05-15 08-47-06\CPK2015-515-04465.D)1.362 1.462 1.8372.110=====================================================================Area Percent Report=====================================================================Sorted By : SignalMultiplier : 1.0000Dilution : 1.0000Do not use Multiplier & Dilution Factor with ISTDsSignal 1: DAD1 C, Sig=254,4 Ref=offPeak RetTime Type Width Area Height Area# [min] [min] [mAU*s] [mAU] %----|-------|----|-------|----------|----------|--------|1 1.362 MM 0.0528 1.80688 5.70622e-1 0.03642 1.462 MM 0.0554 4955.89258 1490.00452 99.88903 1.837 MM 0.0677 2.64214 6.50008e-1 0.05334 2.110 MM 0.0641 1.05738 2.74959e-1 0.0213Totals : 4961.39899 1491.50011=====================================================================*** End of Report ***===========================================================Acq. Operator : Li Shan(LCMS-02) Seq. Line : 50Acq. Instrument : HY-LCMS-02 Location : P1-C-01Injection Date : 5/15/2015 12:20:58 PM Inj : 1Inj Volume : 3.000 µlAcq. Method : D:\AGLIENT 1260\DATA\20150515\20150515 2015-05-15 08-47-06\100-1000MS+3MIN-1.5_(0.02%FA).MLast changed : 5/15/2015 8:47:06 AM by Li Shan(LCMS-02)Analysis Method : D:\AGLIENT 1260\METHOD\BL30-90A,210NM(RP-HPLC).MLast changed : 5/15/2015 3:14:15 PM by Li Shan(LCMS-02)(modified after loading)M ethod Info : HPLC Catalog No : HY-13202 Batch#04465 A-RP-132Additional Info : Peak(s) manually integratedmin 0.51 1.52 2.530100000200000300000400000500000600000700000800000MSD1 TIC, MS File (D:\AGLIENT 1260\DATA\20150515\20150515 2015-05-15 08-47-06\CPK2015-515-04465.D) ES-API, Pos, Sc1.466MS Signal: MSD1 TIC, MS File, ES-API, Pos, Scan, Frag: 50 Spectra averaged over upper half of peaks. Noise Cutoff: 1000 counts. Reportable Ion Abundance: > 10%.Retention Mol. WeightTime (MS) MS Area or Ion1.466 4473754 280.00 I279.00 I278.00 Im/z 100200300400500600020406080100*MSD1 SPC, time=1.434:1.507 of D:\AGLIENT 1260\DATA\20150515\20150515 2015-05-15 08-47-06\CPK2015-515-04465.D ES-API Max: 432781279.0 278.0 *** End of Report ***。

HT-2157_DataSheet_MedChemExpress

Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:HT–2157 (SNAP 37889) is a selective, high–affinity, competitive antagonists of galanin–3 receptor (Gal 3).IC50 & Target: Galanin–3 receptor [1]In Vitro: HT–2157 (SNAP 37889) binds with high affinity to membranes from transiently transfected LMTK – cells expressing the human Gal 3 receptor (K i =17.44±0.01 nM; n>100) and is highly selective for Gal 3 over the Gal 1 and Gal 2 subtypes (K i >10,000 nM for each subtype; n=46 of each subtype). When tested for the antagonism of galanin–evoked inhibition of adenylyl cyclase, HT–2157 (0.1–10μM) produces concentration–dependent rightward shifts of the concentration–effect curve to galanin [1].In Vivo: The galanin–3 receptor antagonist, HT–2157 (SNAP 37889), reduces operant responding for ethanol in alcohol–preferring rats. The novel selective GALR3 antagonist, HT–2157, to reduce anxiety–like behaviour and voluntary ethanol consumption in the iP (alcohol–preferring) rat. Male iP rats treated with HT–2157 at a dose of 30 mg/kg (i.p.) do not show altered locomotor activity or changes in anxiety–like behaviour in the elevated plus maze or light–dark paradigms. Treatment with HT–2157 (30 mg/kg, i.p.)reduces operant responding for solutions containing ethanol, sucrose and saccharin. Collectively, results from the current study shows that HT–2157 (30 mg/kg, i.p.) is effective in reducing operant responding for ethanol, independent of a sedative effect [2].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[1]Binding affinities for HT–2157 (SNAP 37889) and SNAP 398299 at the human Gal 1, Gal 2, and Gal 3 receptors are determined by using the 125I–galanin displacement assay. Additionally, is tested for binding in a broad cross–reactivity panel that included G–protein–coupled receptors, ion channels, enzymes, and transporters. The ability of HT–2157 to antagonize functional responses to galanin is examined in modified HEK–293 cells (PEAK rapid cells) transiently cotransfected with the Gal 3 receptor and Gαz by measuring the inhibition of adenylyl cyclase activity [1].Animal Administration: HT–2157 (SNAP 37889) is prepared in 5% DMSO and 1% HMC in saline [2].[2]Rat [2]The effect of HT–2157 on locomotor activity is examined using adult male iP rats (n=12; 418–467 g). Locomotor activity is tested to ensure that any potential change in anxiety or ethanol consumption in subsequent tests is not due to any sedative property of the drug. Rats are habituated to the locomotor cells (26×26×40 cm) for 60 minute sessions which are conducted daily for threeconsecutive days. To minimise the effect of habituation during the treatment phase, rats are divided into two even groups which received a single injection of either HT–2157 (30 mg/kg, i.p.) or vehicle (1 ml/kg i.p.) 30 min prior to being placed in the locomotor cells for a 60 minute session. The following day the treatments are reversed so that all rats received treatment with both the vehicle and SNAP 37889 compound. Movements in terms of number of moves, move time (s) and total distance travelled (cm), are recorded automatically using TruScan 2.0 software.References:Product Name:HT–2157Cat. No.:HY-100717CAS No.:303149-14-6Molecular Formula:C 21H 13F 3N 2O Molecular Weight:366.34Target:Neuropeptide Y Receptor Pathway:GPCR/G Protein Solubility:DMSO: ≥ 29 mg/mL[1]. Swanson CJ, et al. Anxiolytic– and antidepressant–like profiles of the galanin–3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299. Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17489–94.[2]. Ash BL, et al. The galanin–3 receptor antagonist, SNAP 37889, reduces operant responding for ethanol in alcohol–preferring rats. Regul Pept. 2011 Jan 17;166(1–3):59–67.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

SKIDA 4 Plus 多通道注射器分析仪说明书

Key features• Tests up to four infusion pumps simultaneously• Compatible with virtually any type of infusion device • Instantaneous and average flow measurement • Occlusion pressure measurements to 45 psi • Single- and dual-flow (piggyback) testing• Full PCA testing (bolus volume, lockout time, and automated external triggering)• Autostart mode enables unit to begin testing only when fluid is detected • On-board graphing of pressure and flow• Built-in memory to save test results for printing or downloading to computer • Hydrograph graphical software to control unit and display results via PC • Automated testing through Fluke Biomedical medTester 5000C (US only)Technical DataIDA 4 Plus Multi-Channel Infusion Device Analyzer maximizes productivity with multiple, independent channels for testing up to four infusion pumps at once.The device measures instantaneous flow, average flow, occlusion pressure, and analyzes patient-control analgesia (PCA) units. An optional PCA trigger box provides automated PCA pump control, allowing technicians to set up tests and walk away.An autostart feature simplifies syringe pump testing or other tests that have long startup times.With built-in memory, the IDA 4 Plus records test results internally and provides easy-to-read test-result graphs right on the analyzer’s screen. The display is so large numbers can be read from across the room.Additionally, the IDA 4 Plus comes withHydrograph PC software for creating full-color graphs and reports. For automated testing, the IDA 4 Plus is compatible with Fluke Biomedical’s medTester 5000C (US only).ElsoPhilipsService,Jilemnického2;91101Trenèín;tel:+421326582410,7431690;fax:+421326582592;email:**************;web:www.fluke.skTechnical specificationsFlow-rate measurementTechnique: Calculated by mea-suring a volume over timeRange: 0.5 ml/hr to 1000 ml/hr Accuracy: 1 % of reading ± 1 LSD for flows of 16 ml/hr to 200 ml/hr for volumes over20 ml; otherwise, 2 % of reading ± 1 LSD after delivery of 10 ml Volume measurementTechnique: Volume measured directly by the transducer in minimum sample sizes of 60 µl Range: 0.06 ml to 9999 ml Accuracy: 1 % of reading ± 1 LSD for flows of 16 ml/hr to 200 ml/hr for volumes over20 ml; otherwise, 2 % of reading ± 1 LSD after delivery of 10 ml PCA bolus measurementTechnique: Volume is measured directly by the transducer in minimum bolus volumes of 0.5 ml. The measurement is made with a continuous rate between 0 ml/hr and 30 ml/hr. The bolus flow rate should be at least four times the basal flow rate for reliable detection of boluses.Minimum bolus volume: 0.5 ml Accuracy: See volume measurementPressure measurementTechnique: Direct occlusion of the infusion line and measure-ment of pressure prior to the glass transducerRange: 0 psi to 45 psi and equivalents in mmHg and kPa Accuracy: 1 % of full scale ± 1 LSDBack pressure: -100 mmHg to 300 mmHgElectrical specificationsSupply voltage: Autoswitching90 V ac to 260 V acSupply frequency: 50 Hz to 60 HzSupply power: < 30 VAFuse: 20 mm 250 V, 1 A (T) (slow blow)Earth leakage current: < 1 mA in single fault condition Environmental conditionsOperating temperature: 15 ˚C to 30 ˚C (59 ˚F to 86 ˚F)2 Fluke Biomedical IDA 4 Plus Multi-Channel Infusion Device AnalyzerStorage temperature: 0 ˚C to 40 ˚C (32 ˚F to 104 ˚F) at 85 % RH or less for storage [Do not leave for more than 48 hours at -20 ˚C (-4 ˚F)]General informationDimensions (WxDxH): 19.05 cm x 18.11 cm x 30.18 cm (7.5 in x 7.2 in x 11.9 in)Weight: 5 kg (11 lb)“demand” signals. With the IDA 4 Plusconnected to the Trigger Box, it will measure,display, and record essential PCA pumpparameters to show bolus and flow characteristics.The PCA Trigger Box can also be used to test the “nurse call” interface of PCA Pumps. Simulate “nurse calls” and look for the response indicated by makers in the flow graph.HydroGraph™Graphics SoftwareUse the moving colorvisual advantage ofHydroGraph to troubleshootup to four infusion pumpsat once. Data is takendirectly off the transducerand transmitted to HygroGraph.The flowing graphs provide anelectronic means to display, store, and recall flow patterns for comparison at a later date. Each test window can display instantaneous and average flow rates, cumulative, and bolus volumes; and occlusion pressure.IDA 4 Plus Multi-Channel Infusion Device Analyzer Fluke Biomedical 3Ordering informationModelsIDA 4 Plus One-Channel Infusion Device Analyzer 2250063 IDA-4P/1-US120V (US)2394575 IDA-4P/1-AUS250V(Australia)2394582 IDA-4P/1-DEN250V(Denmark)2394594 IDA-4P1-SHK250V (Shuko)2394608 IDA-4P/1-ISR250V (Israel)2394613 IDA-4P/1-ITAL250V (Italy)2394624 IDA-4P/1-IND250V (India)2394636 IDA-4P/1-SWZ250V(Switzerland)2394649 IDA-4P/1-UK250V (UK) IDA 4 Plus Two-Channel Infusion Device AnalyzerFull testing for up to two infusion pumps simultaneously2250088IDA-4P/2-US120V (US)2394651IDA-4P/2-AUS250V(Australia)2394660IDA-4P/2-DEN250V(Denmark)2394672IDA-4P/2-SHK250V (Shuko)2394685IDA-4P/2-ISR250V (Israel)2394697IDA-4P/2-ITAL250V (Italy)2394703IDA-4P/2-IND250V (India)2394715IDA-4P/2-SWZ250V(Switzerland)2394726IDA-4P/2-UK250V (UK)IDA 4 Plus Three-ChannelInfusion Device AnalyzerFull testing capability for up to threeinfusion pumps simultaneously2250109IDA-4P/3-US120V (US)2394732IDA-4P/3-AUS250V(Australia)2394744IDA-4P/3-DEN250V(Denmark)2394759IDA-4P/3-SHK250V (Shuko)2394767IDA-4P/3-ISR250V (Israel)2394771IDA-4P/3-ITAL250V (Italy)2394780IDA-4P/3-IND250V (India)2394798IDA-4P/3-SWZ250V(Switzerland)2394800IDA-4P/3-UK250V (UK)IDA 4 Plus Four-ChannelInfusion Device AnalyzerFull testing capability for up to fourinfusion pumps simultaneously2250127IDA-4P/4-US120V (US)2394817IDA-4P/4-AUS250V(Australia)2394821IDA-4P/4-DEN250V(Denmark)2394839IDA-4P/4-SHK250V (Shuko)2394842IDA-4P/4-ISR250V (Israel)2394856IDA-4P/4-ITAL250V (Italy)2394863IDA-4P/4-IND250V (India)2394874IDA-4P/4-SWZ250V(Switzerland)2394888IDA-4P/4-UK250V (UK)Standard accessories2213506Electronic User Manual andHydroGraph Software2238626Serial Communication Cable221723120 ml priming syringe2391750Luerlock-3 way (one foreach channel)2238909 5 foot plastic drain lineXXXXXXX Power cord (countryspecific)Optional accessories2245061 External Mini-Keyboard,83-Key with PS/2Connector and AT Adapter2238072 Parallel Printer Cable(D25M-36M)2209703 PCA Trigger/Nurse Call Box2248899 Printer, Seiko DPU-414-30B(120 V power supply)(additional purchaserequired: Parallel PrinterCable, p/n 2238072)2399531 Printer, Seiko DPU-414-30B(220 V power supply)(additional purchaserequired: Parallel PrinterCable, p/n 2238072)2235375 Printer (120 V powersupply)2235382 Printer (220 V powersupply)2200102 Interface Cable, medTesterto IDA 4 Plus (withoutwedge adapter) (RS-232;female DB25 to female DB9)2201042 Interface Cable, medTesterto IDA 4 Plus (with wedgeadapter) (RS-232; femaleDB9 to female DB25)2245092 Barcode Scanner (withlong-reach coil cable withY connector for keyboardattachment)2238626 Null Modem Cable (femaleDB9 to female DB9)Fluke Biomedical.Better products. More choices. One company.Fluke Biomedical 6045 Cochran RoadCleveland, OH 44139-3303 U.S.A.Fluke Biomedical Europe Science Park Eindhoven 5110, 5692EC Son, The NetherlandsFor more information, contact us:In the U.S.A. (800) 850-4608 or Fax (440) 349-2307In Europe/M-East/Africa +31 40 267 5200 or Fax +31 40 267 5436From other countries +1 (440) 248-9300 or Fax +1 (440) 349-2307Email:*************************Web access: ©2006-2008 Fluke Biomedical. Specifications subject to change without notice. All OEM trademarks are implied. Printed in U.S.A. 3/2008 2804429 D-EN-N Rev Briešenia na presné meranie™Elso Philips Service; tel: +421 32 6582410email: ************; web: www.elso.sk。

PrepSEQ Nucleic Acid Extraction Kit使用手册说明书

PrepSEQ ™ Nucleic Acid Extraction KitTotal Nucleic Acid (DNA and RNA) ExtractionCatalog Numbers 4480466 and 4428176Pub. No. MAN0019336 Rev. C.0Product descriptionThe PrepSEQ ™Nucleic Acid Extraction Kit is designed for preparation of high-quality total nucleic acid (NA) from tissue, liquid, and swab samples. Magnetic beads allow efficient DNA and RNA capture and sample washing.This user guide describes the following methods:•“Isolate total nucleic acid from tissue samples” on page 2•“Isolate total nucleic acid from liquid samples” on page 3•“Isolate total nucleic acid from swab samples (manual method)” on page 4•“Isolate total nucleic acid from swab samples (automated method with the KingFisher ™ mL Food Protection Purification System)” on page 5Kit contents and storage[1]Refer to the product label for the expiration date.[2]Add ~35 mL of 100% isopropanol to the empty bottle before use.[3]Add 74 mL of 95% ethanol before use.Note: Kit components may ship separately depending on configuration and storage conditions.Required materials not suppliedUnless otherwise indicated, all materials are available through . "MLS" indicates that the material is available from or another major laboratory supplier.Isolate total nucleic acid from tissue samplesa.Place up to 100 mg of solid (tissue) sample in a 1.5-mL microcentrifuge tube.b.Add 300 μL of PK Buffer and 10 μL of Proteinase K.c.Incbate for 60 minutes at 45℃ and 1000 rpm in the thermomixer.d.Centrifuge for 2 minutes ar 10,000 x g , then transfer the supernatant to a new 1.5-mL centrifugetube.e.Add 200 μL of Lysis Buffer, then vortex for 15 seconds.1Treat the samples with proteinase K and perform cell lysisVortex the Magnetic Particles until complete resuspension (approximately 5 seconds).a.Add 35 μL of Magnetic Particles to the sample.b.Vortex for 10 seconds at low speed.c.Add 350 μL of Binding Solution, then vortex for 5 seconds.d.Incubate for 10 minutes at room temperature shaking continuously.e.Vortex for 10 seconds at low speed, then place the tube in the DynaMag ™‑2 Magnet.f.Let the tube rest in the DynaMag ™‑2 Magnet until complete separation occurs (approximately 1-2minutes).g.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.2Bind the nucleic acid to the magnetic beadsa.Add 300 μL of Wash Solution to the tube, then vortex at medium speed for 5 seconds, or untilthe pellet is completely resuspended.b.Place the tube in the DynaMag ™‑2 Magnet, then let it rest for 30 seconds.c.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.d.Repeat the two last steps two more times.3Wash the nucleic acida.Air-dry the Magnetic Particles in the DynaMag ™‑2 Magnet with the lid open for 5 minutes.b.Add 50 μL of Elution Buffer.c.Close the lid, then vortex the tube at medium speed for 5 seconds.d.Incubate the tube for 5 minutes at 45℃.e.Vortex the tube at medium speed for 2 seconds, then place the tube in the DynaMag ™‑2 Magnet.f.Let the tube rest in the DynaMag ™‑2 Magnet for at least 1 minute.g.Transfer the liquid phase containing the total NA to a new tube for storage.4Elute the nucleic acid Isolate total nucleic acid from liquid samplesa.Place 250 μL of liquid sample in a 1.5-mL microcentrifuge tube.b.Add 50 μL of PK Buffer and 10 μL of Proteinase K, then vortex for 15 seconds.c.Incbate for 25 minutes at 45℃ and 1000 rpm in the thermomixer.d.Centrifuge for 2 minutes ar 10,000 x g , then transfer the supernatant to a new 1.5-mL centrifugetube.e.Add Lysis Buffer up to 500 μL of total volume, then vortex for 15 seconds.1Treat the samples with proteinase K and perform cell lysisVortex the Magnetic Particles until complete resuspension (approximately 5 seconds).a.Add 35 μL of Magnetic Particles to the sample.b.Vortex for 10 seconds at low speed.c.Add 350 μL of Binding Solution, then vortex for 5 seconds.d.Incubate for 10 minutes at room temperature shaking continuously.e.Vortex for 10 seconds at low speed, then place the tube in the DynaMag ™‑2 Magnet.f.Let the tube rest in the DynaMag ™‑2 Magnet until complete separation occurs (approximately 1-2minutes).g.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.2Bind the nucleic acid to the magnetic beadsa.Add 300 μL of Wash Solution to the tube, then vortex at medium speed for 5 seconds, or untilthe pellet is completely resuspended.b.Place the tube in the DynaMag ™‑2 Magnet, then let it rest for 30 seconds.c.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.d.Repeat the two last steps two more times.3Wash the nucleic acida.Air-dry the Magnetic Particles in the DynaMag ™‑2 Magnet with the lid open for 5 minutes.b.Add 50 μL of Elution Buffer.c.Close the lid, then vortex the tube at medium speed for 5 seconds.d.Incubate the tube for 5 minutes at 45℃.e.Vortex the tube at medium speed for 2 seconds, then place the tube in the DynaMag ™‑2 Magnet.f.Let the tube rest in the DynaMag ™‑2 Magnet for at least 1 minute.g.Transfer the liquid phase containing the total NA to a new tube for storage.4Elute the nucleic acid Isolate total nucleic acid from swab samples (manual method)a.Place the swab sample in a 1.5-mL microcentrifuge tube.b.Add 650 μL of Lysis Buffer, then vortex for 15 seconds.c.Incbate for 25 minutes at 45℃ and 1000 rpm in the thermomixer.d.Centrifuge for 2 minutes ar 10,000 x g , then transfer 500 μL of supernatant to a new 1.5-mLcentrifuge tube.1Perform cell lysis Vortex the Magnetic Particles until complete resuspension (approximately 5 seconds).a.Add 35 μL of Magnetic Particles to the sample.b.Vortex for 10 seconds at low speed.c.Add 350 μL of Binding Solution, then vortex for 5 seconds.d.Incubate for 10 minutes at room temperature shaking continuously.e.Vortex for 10 seconds at low speed, then place the tube in the DynaMag ™‑2 Magnet.f.Let the tube rest in the DynaMag ™‑2 Magnet until complete separation occurs (approximately 1-2minutes).g.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.2Bind the nucleic acid to the magnetic beadsa.Add 300 μL of Wash Solution to the tube, then vortex at medium speed for 5 seconds, or untilthe pellet is completely resuspended.b.Place the tube in the DynaMag ™‑2 Magnet, then let it rest for 30 seconds.c.Carefully discard the liquid phase without disturbing the Magnetic Particles pellet.d.Repeat the two last steps two more times.3Wash the nucleic acida.Air-dry the Magnetic Particles in the DynaMag ™‑2 Magnet with the lid open for 5 minutes.b.Add 50 μL of Elution Buffer.c.Close the lid, then vortex the tube at medium speed for 5 seconds.d.Incubate the tube for 5 minutes at 45℃.e.Vortex the tube at medium speed for 2 seconds, then place the tube in the DynaMag ™‑2 Magnet.4Elute the nucleic acid4Elute the nucleic acid (continued)f.Let the tube rest in the DynaMag™‑2 Magnet for at least 1 minute.g.Transfer the liquid phase containing the total NA to a new tube for storage.Isolate total nucleic acid from swab samples (automated method with the KingFisher™ mL Food Protection Purification System)For more information about using the KingFisher™ mL Food Protection Purification System, see Thermo Scientific™ KingFisher™ mL User Manual (Pub. No. 1508260).•Ensure that the PSNA_mL_300ul script has been downloaded from the product page and loadedonto the KingFisher™ mL Food Protection Purification System.•Ensure that a water bath or heating block is heated to 83°C.•Label the following consumables for each sample to be processed and the negative extractioncontrol:–One tube strip–Two 1.5‑mL microcentrifuge tubes (nuclease free)Note: Up to 14 samples and 1 negative extraction control can be processed at a time on theKingFisher™ mL Food Protection Purification System.1Before you beginVortex the Magnetic Particles until complete resuspension (approximately 5 seconds).a.For the number of required reactions, prepare the Binding Mix according to the following table:[1]Include 10% overage when making for multiple reactions.b.Invert the Binding Mix 5 times gently to mix, then add 700 µL to Tube A of each tube strip.Include tube strips for each sample and negative extraction control.Note: Remix the Binding Mix by inversion frequently during pipetting to ensure even distributionof beads to all samples or wells. The Binding Mix is viscous, so pipet slowly to ensure that thecorrect amount is added. DO NOT reuse pipette tips to add Binding Mix to the samples, as thehigh viscosity will cause variations in the volumes added.c.Add 300 µL of Wash Buffer to Tube B and 300 µL of Wash Buffer to Tube C of each tube strip.d.Add 100 µL of Elution Buffer to Tube D of each tube strip.e.Add 1 µL of Total RNA Control (Human) to Tube A of each tube strip.f.Vortex the swab sample tubes for 30 seconds.g.Add 300 µL of a sample to Tube A of the corresponding, pre‑labeled tube strip. Repeat for theremaining samples and tube strips.h.Add 300 µL of Nuclease-free Water (not DEPC-Treated) to Tube A of the Negative ExtractionControl tube strip.2Set up processing tubesa.Load the prepared tube strips into the tray, then place the tray in the KingFisher ™mL FoodProtection Purification System.b.Fully insert the tip combs into the tip comb slots.c.Select the PSNA_mL_300ul script, then press Start .d.When prompted by the instrument, remove the tube ‑strip tray from the instrument.e.For each tube strip, transfer the elution buffer (100 µL) from Tube D into one of thecorresponding pre ‑labeled microcentrifuge tubes.f.Cap the microcentrifuge tubes, then incubate at 83°C for 4 minutes.g.Transfer the elution buffer from each microcentrifuge tube back into Tube D of thecorresponding tube strip.h.Load the tube ‑strip tray into the instrument, then restart the run.i.After the run is complete, immediately remove the tube ‑strip tray from the instrument.j.For each tube strip, transfer the elution buffer (100 μL) from Tube D into the second pre ‑labeled microcentrifuge tube.Place the microcentrifuge tubes on ice for immediate use in real-time PCR. The extracted samples can be stored at -70°C for long ‑term storage (up to one year).3Process the samples on the instrumentLimited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies' General Terms and Conditions of Sale at /us/en/home/global/terms-and-conditions.html . If you have any questions, please contact Life Technologies at /support .Life Technologies Ltd | 7 Kingsland Grange | Woolston, Warrington WA1 4SR | United KingdomFor descriptions of symbols on product labels or product documents, go to /symbols-definition .The information in this guide is subject to change without notice.DISCLAIMER : TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.Important Licensing Information : These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.©2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. Thermomixer ™is a trademark of Eppendorf./support | /askaquestion 。

微化学Ti xLiftx图像反转胶类技术数据表说明书

TI xLiftximage reversal resi Technical Data Sheetrevised 01/2018General InformationThe TI xLiftx image reversalre (5 .. 20 µm) films. It is the succe almost identical, the difference isThis technical data sheet intends for various applications. Howev exposure dose, or development d adjusted on each individual demam resistal resist is optimized for lift-off of thick successor of the former TI xLift resist. Both ce is a minor change in the ingredients. tends to give you a guide-line for process p owever, the optimum values for e.g. sp ent depend on the individual equipment and demand.←10.5 µm thick metal via lift-off witMicroChemicalsNicolaus-Otto D-89079 Ulm Fon +49 (0) 731 97Mobil +49 (0) 178 78Fax +49 (0) 731 97Email info@microchem Internetwww.microchemmk to very thick Both resists are ess parameters . spin profile, and need to bef with TI xLiftx icals GmbHtto-Str. 39 lm 977343 0 8 7825198 977343 29 'Image Reversal' – A Short IntroductionWhat 'image reversal' generally meansinverted mask (the exposed areas finally remain)behaves like an exposed positive resist.cross-links the exposed area, while the unexposed area remains photo-active(without mask) ...which was not exposed in the first step, soluble in developerareas exposed in the first step now remain... and for what image reversal is good for: Adjustable undercut for lift-off of thin and thick sputtered, CVD, and evaporated films like metals, a-Si:H, a-SiN:H etc.→Processing the TI xliftxIn chronological order:■After cleaning the substrate, put the substrate on the hotplate at minimum 120 °C for 10 minutes to remove adsorbed water from the substrate surface.Alternatively, you can use a furnace at same temperature for 30 min. Standard HMDS procedure (only from vapor phase with an optimum substrate temperature of 125°C !) is also an adequate preparation.■Spin-coat the resist immediately after cooling down the substrate at: resist thickness 5.9 µm 7.0 µm 8.2 µm 10.5 µm 14.4 µm 20.0 µm final rpm 2.200 1.600 1.400 1.200 900 600 with an acceleration of approx. 900 rpm/s and keep at final spin speed for 5 seconds. Then decelerate down to 0 rpm at approx. –900 rpm/sec. For 4 inch wafer, a minimum of 3.0 ml of resist is recommended.■Leave the substrate on the spin-coater (or on any horizontal surface at room temperature) for at least 5 min (≈5 µm resist thickness) to 30 min (≈20 µm resist thickness). This will smooth the surface, reduce striations, and allow the edge bead near the substrate edge i) to flatten and ii) to contract to the outermost few mm of the wafer. If subsequent soft-bake causes foaming or patterning of the resist, increase the delay.■Soft-bake at 95°C on a hot-plate or in an fufor:total resist thickness 5 .. 9 µm 10 .. 16 µm 17 .. 24 µm time (min) 20 40 60■Remove the edge bead on the edge of the substrate. We recommend giving a sharp stream of AZ EBR solvent (PGMEA) focused on the outer few mm of the substrate spinning at 1.000 rpm. Avoid spattering drops of EBR on the inner side of the substrate, this will cause spots with reduced resist thickness.■Exposure broadband or i-line (with the mask) at a (as calibrated on i-line, 365 nm) dose of approximately:total resist thickness 5.9 µm 7.0 µm 8.2 µm 10.5µm 14.4µm20.0µmfirst exposure dose (mJ/cm2) 40 ..10050 ..14080 ..200110 ..280150 ..400180 ..500= exposure time (sec.)(holds for all standard maskaligners with a 350W Hg-lamp)4 .. 105 .. 14 8 .. 20 11 .. 28 15 .. 40 18 .. 50This first exposure dose adjusts the final wall profile and can be increased/decreased by up to 50% for optimum results (see Appendix).■Post-exposure delay time:total resist thickness 5 .. 9 µm 10 .. 16 µm 17 .. 24 µm Room temperature 20° (min) ... 5 5 10... subsequent hot-plate 50°C (min) 0 15 30In this delay time, N 2, generated during exposure, will diffuse out the resist. If the resist foams on the hot-plate, next time increase the delay time at room temperature.■ Reversal Bake: After the delay bake the substrate at a temperature of 130°Con the hotplate for 2 minutes (when using furnace try 20 minutes at 120°C-130°C. Because this step is very temperature critical furnace baking is not recommended). This step is the reversal bake where the image is reversed due to cross link the exposed areas making them insoluble in the developer. If the resist foams, next time increase the delay time at 50°C (see previous point) ■ Flood Exposure: Exposure the substrate for the second time without a mask(flood exposure).total resist thickness 5 .. 9 µm 10 .. 16 µm 17 .. 24 µm dose (mJ/cm 2)≈ 800≈ 1.000≈ 1.200When, during a subsequent deposition, the temperature will raise over 80°C, use a high exposure dose to avoid nitrogen bubbles in the resist during the deposition. ■Develop e.g. in AZ 826MIF. When the structures are through-developed, add another approx. 10-30% of the time in the bath of development time to finalize the side wall profile (see appendix). Beside the used developer, development time depends on resist thickness and strongly on reversal bake temperature. If shorter development times are important, use AZ 400K 1:3.5 (e.g. 100 ml AZ 400K and 350 ml DI-water). Resist dark erosion might slightly increase.600900120015001800210046810121416182022r e s i s t t h i c k n e s s (µm )spin speed (rpm)Appendix A: Adjusting the undercutUndercut depends on:1.First exposure doseAt low 1st exposure doses, only the upper part of the resist is exposed. Only this region will subsequently be 'reversed' during image reversal bake. The substrate-near resist now is still unchanged and will be exposed (and made soluble) in the subsequent flood exposure. If the first exposure dose is too low, development will lift the upper resist layer, or/and the dark erosion is too high.→ High 1st exposure dose: Reduced undercut2.Reversal bake temperature and reversal bake timeIf the reversal bake temperature- and time is too low, the image reversal process will not be completed. As a consequence, the resist exposed during the 1st exposure shows a high 'dark erosion' during development, sometimes appearing as holes or bubbles in the resist. With increasing reversal bake temperature/-time, the reversal process improves, and dark erosion is reduced.At reversal bake temperatures/-times chosen to high, even the parts of the resists exposed by scattered light during the 1st exposure (in regions where no light should be) are reversed. As a consequence, the profile might change from the desired undercut to a typical positive profile.→ High reversal bake temperature/-time: Reduced undercut3.Developer/Development timeWith increased developing time, or by the usage of strong developers (like AZ 400K in dilutions of 1:3.5 and stronger), also the parts of the resist are developed, where the image reversal process has been performed.→ High developing time: Increased undercutAppendix B: ParametersExample I: Varying the first exp(Thickness 12 µm; Rev. bake 130°C 2m1st exp. 85 mJ/cm2170 mExample II: Varying the develo(Thickness 15 µm; 1st exp. 240 mJ, RevDev. time 5.0 min6.5 mExample III: Varying the devel(Thickness 13 µm; 1st exp. 310 mJ, RevDev. time 5.0 minExample IV: Varying the revers(Thickness 11 µm; Rev. bake 130°C; Florev. bake time 1 min11 mueeters and profilet exposure dose at 30% 'over-developing'C 2min; Flood exp. 1.000 mJ; Dev. AZ400K 1:3, 8 min170 mJ 260 mJ 430velopment time at low first exposure dose, Rev. bake 130°C 2min; Flood exp. 1.200 mJ; Dev. A6.5 min 8.5 min 15evelopment time at high first exposure dos, Rev. bake 130°C 2min; Flood exp. 1.200 mJ; Dev. Aversal bakeC; Flood exp. 1.000 mJ; Dev. AZ 826MIF)3 min 6 minng'min)430 mJoseev. AZ400K 1:2.5)15 mindoseev. AZ400K 1:2.5)min。

VX-809_936727-05-8_DataSheet_MedChemExpress

Product Name:VX-809CAS No.:936727-05-8Cat. No.:HY-13262Product Data SheetMWt:452.41Formula:C24H18F2N2O5Purity :>98%Solubility:DMSO ≥90mg/mL Watery Mechanisms:Biological Activity:VX 809acts to correct CFTR mutations common in cystic fibrosis by increasing mutant CFTRPathways:Membrane Transporter/Ion Channel; Target:CFTR g <1.2mg/mL Ethanol ≥4.6mg/mLVX-809 acts to correct CFTR mutations common in cystic fibrosis by increasing mutant CFTR(F508del-CFTR) maturation,EC50 of 0.1 μM.IC50 value: 0.1 uM (EC50)Target: CFTR VX-809 is the second investigational oral candidate compound for the treatment of cystic fibrosis (CF). VX-809 may act to restore the function of the cystic fibrosis transmembrane conductanceregulator (CFTR) protein, the defective cell membrane protein responsible for the progression of CF.VX-809 defects in the CFTR protein affect the transport of chloride and other ions across cells, and lead to the accumulation of thick, sticky mucus in the lungs of patients with CF. This mucus fosters References:[1]. Clancy JP, et al. Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thoraxdoi:10.1136,y g pchronic infection and inflammation, and results in irreversible lung damage....[2]. Pettit RS. Cystic fibrosis transmembrane conductance regulator-modifying medications: thefuture of cystic fibrosis treatment.Ann Pharmacother. 2012 Jul;46(7-8):1065-75. Epub 2012 Jun 26.[3]. Lubamba B, et al. Cystic fibrosis: Insight into CFTR pathophysiology and pharmacotherapy.ClinBiochem. 2012 Jun 12.[4]. Van Goor F, et al. Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18843-8. Epub 2011Oct 5.[5]. Kim Chiaw P, et al. Insights into the mechanisms underlying CFTR channel activity, the molecular basis for cystic fibrosis and strategies for therapy Caution: Not fully tested. For research purposes onlyMedchemexpress LLCmolecular basis for cystic fibrosis and strategies for therapy...18 W i l k i n s o n W a y , P r i n c e t o n , N J 08540,U S AE 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。

维泰克2客户信-clsi折点性能参数补充说明书

VITEK 2客户信编号: CSN 4757-2尊敬的生物梅里埃用户：2020年7月, 生物梅里埃发布了客户信CSN-4757通知VITEK®2用户梅里埃已经完成了气单胞菌和弧菌多个抗生素CLSI折点的验证工作。

如果您在2020年7月收到通知后已经更新了折点, 我们抱歉地通知您当时发布的客户信表2性能参数部分存在笔误。

此次客户信的表2已经进行了更新，为便于识别，更新部分用黄色高亮显示。

该客户信其它部分同此前版本保持一致。

该评估使终端用户可以在VITEK® 2软件中应用CLSI折点。

表1中的CLSI折点都已经成功验证，均满足可接受标准，可用于患者或其它菌株测试，某些情况下需要增加一些限制信息.为了应用这些折点，需要在AES中创建自定义参数。

请参考VITEK® 2软件用户手册（修改折点部分）中的详细信息。

这些折点会被整合进未来新版本VITEK® 2软件Global CLSI-based参数集。

表2提供了应用CLSI折点下这些抗菌药物的性能参数信息。

之前出版的VITEK® 2文档尚未公布这些性能参数信息。

假如您选择实施这些CLSI折点，请留存该客户信。

附录1：举例如何创建高级报告工具(BIOART)规则，用于管理该客户信5-8页中的局限。

做为我们持续改善项目的一部分，梅里埃致力于提升产品质量，功能和易用性。

感谢您选择使用VITEK® 2系统，如果您有任何问题，请随时联系您当地的生物梅里埃代表。

此致敬礼梅里埃诊断产品（上海）有限公司2022年5月10日bioMérieux (Shanghai) Company Limited/梅里埃诊断产品(上海)有限公司表1:使用更新后的CLSI折点，表格中抗菌药物性能满足要求bioMérieux (Shanghai) Company Limited/梅里埃诊断产品(上海)有限公司*以下抗生素/细菌组合的结果存在局限，需要屏蔽报告。

pH响应性磁性介孔纳米载药系统的制备

化工进展Chemical Industry and Engineering Progress2023 年第 42 卷第 10 期pH 响应性磁性介孔纳米载药系统的制备刘慧慧，史笑飞，王倩男，刘锦博，张静（郑州大学化工学院，河南郑州 450001）摘要：研究开发了一种基于磁性介孔二氧化硅和聚多巴胺（PDA ）混合体的智能给药平台，可用于pH 响应性给药。

通过共沉淀法制备Fe 3O 4，以十六烷基三甲基溴化铵（CTAB ）为模板，硅酸四乙酯（TEOS ）为硅源，通过Stober 法获得了磁性纳米材料Fe 3O 4@mSiO 2。

负载抗癌药物阿霉素（DOX ）后，通过碱性条件下多巴胺的氧化自聚合，在其表面形成pH 敏感的PDA 涂层，得到DOX/Fe 3O 4@mSiO 2@PDA 磁性纳米载药系统。

通过透射电子显微镜、X 射线衍射、傅里叶红外光谱仪、比表面积测试和振动样品磁强计等技术手段对材料进行表征。

结果表明Fe 3O 4@mSiO 2的形貌为球形，拥有良好介孔结构和高达619.16m 2/g 的比表面积；VSM 表明载体Fe 3O 4@mSiO 2@PDA具有良好的磁性。

当载药过程中DOX 浓度为0.5mg/mL 、温度为37℃，反应36h 后，载药率为51.69%，包封率为82.70%。

释药曲线表明纳米载药系统具有pH 响应性和药物缓释的特点；噻唑蓝比色法（MTT 法）细胞毒性实验表明Fe 3O 4@mSiO 2@PDA 生物相容性良好，DOX/Fe 3O 4@mSiO 2@PDA 有良好的抗肿瘤效果。

该纳米载药系统在药物靶向递送中具有潜在的应用前景。

关键词：四氧化三铁；磁性纳米材料；介孔；pH 响应性；药物递送；阿霉素中图分类号：O614.881 文献标志码：A 文章编号：1000-6613（2023）10-5390-09Preparation of pH responsive magnetic mesoporous nanoparticledrug loading systemLIU Huihui ，SHI Xiaofei ，WANG Qiannan ，LIU Jinbo ，ZHANG Jing(College of Chemical Engineering, Zhengzhou University, Zhengzhou 450001, Henan, China)Abstract: We developed an intelligent drug delivery platform based on the mixture of magnetic mesoporous silica and polydopamine (PDA), which can be used for pH responsive drug delivery. Magnetic cores Fe 3O 4 was prepared by co -precipitation method. The magnetic nanomaterial Fe 3O 4@mSiO 2 was obtained by the Stober method using cetyltrimethylammonium bromide (CTAB) as a template and tetraethyl silicate (TEOS) as a silicon source. Anticancer drug doxorubicin (DOX) was used as the model drug to be encapsulated into Fe 3O 4@mSiO 2. The surface of the carrier was modified with pH-sensitive PDA coating by oxidation self-polymerization of dopamine at alkaline pH to obtain the magnetic nano drug loading system DOX/Fe 3O 4@mSiO 2@PDA. The materials were characterized by Transmission electron microscope, X-ray diffraction, Fourier Transform infrared spectrometer, Brunauer Emmett Teller and vibration sample magnetometer. We found that Fe 3O 4@mSiO 2 was spherical with good mesoporous structure and a specific surface area of up to 619.16m 2/g. VSM indicated that the carrier Fe 3O 4@mSiO 2@PDA had good magnetic properties. During the drug loading process, when DOX concentration was 0.5mg/mL,研究开发DOI ：10.16085/j.issn.1000-6613.2022-2032收稿日期：2022-11-01；修改稿日期：2023-02-01。

基于QuEChERS_样品前处理的间接竞争酶联免疫分析法快速检测薏苡仁中黄曲霉毒素B1

广东药科大学学报Journal of Guangdong Pharmaceutical University Nov,2023,39(6)基于QuEChERS 样品前处理的间接竞争酶联免疫分析法快速检测薏苡仁中黄曲霉毒素B 1张磊1，2，3，关凯仪1，2，3，黄雨心1，2，3，王淑美1，2，3（1.广东药科大学中药学院，广东广州510006；2.国家中医药管理局中药数字化质量评价技术重点研究室，广东广州510006；3.广东省中药质量工程技术研究中心，广东广州510006）摘要：目的解决间接竞争酶联免疫分析法（indirect competitive enzyme-linked immunosorbent assay ，ic-ELISA ）检测薏苡仁中黄曲霉毒素B 1（Aflatoxin B 1，AFB 1）的基质干扰问题，建立一种简便的高通量检测方法，用于薏苡仁中AFB 1的快速筛查。

方法以ic-ELISA 的显色值和抑制率为评价指标优化样品前处理过程，重点考察QuEChERS 萃取净化技术、不同稀释溶剂和稀释方式消除基质效应的效果，并对建立的方法进行方法学考察，最后应用于薏苡仁实际样品检测，检测结果用液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry ，LC-MS/MS ）进行确证。

结果建立了一种无需任何校正，可用溶剂标准曲线进行定量分析的ic-ELISA 。

方法的半数抑制浓度为0.074ng/mL ，线性范围为1.37~20.0μg/kg ，加标回收率在75.12%~84.46%之间，RSD 小于6%。

122批薏苡仁及其相关样品中AFB 1的阳性率为20.5%，阳性样品的超标率为24.0%，污染水平为1.76~27.56μg/kg 。

ic-ELISA 与LC-MS/MS 检测结果的相关系数（r ）为0.98。

结论QuEChERS 萃取净化技术能够有效去除薏苡仁中的干扰成分，对快速样品前处理方法的建立具有重要应用价值，结合本研究所建立的ic-ELISA ，为薏苡仁中AFB 1的检测和监测提供简便有效的技术手段。

VX-809_DataSheet_MedChemExpress

Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:VX–809 is a CFTR corrector, which improves F508del–CFTR maturation with EC 50 of 0.1±0.1 μM.IC50 & Target: EC50: 0.1 μM (CFTR)[1]In Vitro: In fischer rat thyroid (FRT) cells, VX–809 improves F508del–CFTR maturation by 7.1±0.3 fold (n=3) compared withvehicle–treated cells (EC 50, 0.1±0.1 μM; n=3) and enhances F508del–CFTR–mediated chloride transport by approximately fivefold (EC 50, 0.5±0.1 μM; n=3). At VX–809 concentrations greater than 10 μM, the response is reduced, resulting in a bell–shaped dose–response relationship with an IC 50 of approximately 100 μM. VX–809 is orally bioavailable in rats and achieved in vivo plasma levels significantly above concentrations required for in vitro efficacy [1]. VX–809 produces a concentration–dependent increase in the HRP luminescence signal after incubation with cells at 37°C or 27°C in both cell lines, with a similar EC 50 value of approximately 0.3 μM. In ?F508–HRP CFBE41o – cells at 37°C, VX–809 increases the signal maximally to approximately 250 luminescence arbitrary units (a.u.) over the DMSO control baseline of approximately 60 a.u., representing an approximately 4–fold signal increase. Similarly,with the R1070W–HRP CFBE41o – cells, VX–809 increases the signal maximally to approximately 220 a.u. over the DMSO control baseline of approximately 85 a.u., representing an approximately 2.5–fold signal increase. Therefore, both cell lines produced robustsignals with a good dynamic range for high–throughput screening [2].In Vivo: Oral dosing of 1 mg/kg VX–809 in male Sprague–Dawley rats results in a C max of 2.4±1.3 μM with a t 1/2 of 7.7±0.4 h(mean±SD; n=3), indicating that that VX–809 is orally bioavailable and able to reach plasma levels that significantly exceeded EC 50s for F508del–CFTR correction [1].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[2]Screening is carried out using a Beckman Coulter Biomek FX platform. In one set of assays,R1070W–?F508–CFTR–HRP (R1070W–HRP)–expressing CFBE41o – cells are incubated with 100 μL medium containing 25 μM test compounds and 0.5 μg/mL Doxycycline for 24 hours at 37°C. In a second set of assays, ?F508–CFTR–HRP (?F508–HRP)–expressing CFBE41o – cells are incubated with 100 μL medium containing 25 μM test compounds, 2 μM VX–809, and 0.5 μg/mL doxycycline for 24 hours at 37°C. All compound plates contain negative controls (DMSO) and positive controls (2 μM VX–809). In both assays, the cells are washed four times with PBS, and HRP activity is assayed by the addition of 50 μL/well of HRP substrate. After shaking for 5minutes, chemiluminescence is measured using a Tecan Infinite M1000 plate reader equipped with an automated stacker(integration time, 100 milliseconds)[2].Cell Assay: VX–809 is dissolved in DMSO and stored, and then diluted with appropriate medium before use [2].[2].A549 cells expressing ?F508–CFTR YFP are grown at 37°C/5% CO 2 for 18–24 hours after plating. The cells are then incubated with 100 μL of mediumcontaining test compounds for 18–24 hours. At the time of the assay, cells are washed with PBS and then incubated for 10 minutes with PBS containing forskolin (20 μM) and genistein (50 μM). Each well is assayed individually for I – influx by recording fluorescence continuously (200 milliseconds per point) for 2 seconds (baseline) and then for 12 seconds after rapid addition of 165 μL PBS in whichProduct Name:VX–809Cat. No.:HY-13262CAS No.:936727-05-8Molecular Formula:C 24H 18F 2N 2O 5Molecular Weight:452.41Target:CFTR Pathway:Membrane Transporter/Ion Channel Solubility:10 mM in DMSO137 mM Cl– is replaced by I–. The initial I– influx rate is computed by fitting the final 11.5 seconds of the data to an exponential for extrapolation of initial slope, which is normalized for background–subtracted initial fluorescence. All compound plates contain negative controls (DMSO vehicle) and positive controls (5 μM VX–809). Fluorescence is measured using a Tecan Infinite M1000 plate reader equipped with a dual syringe pump (excitation/emission 500/535 nm)[2].Animal Administration: VX–809 is prepared in a vehicle consisting of 0.5% Tween80/0.5% methylcellulose/water[1].[1]Rat[1]Male rats (n=3 per dose group) are orally administered VX–809 in a vehicle consisting of 0.5% Tween80/0.5% methylcellulose/water at a dose volume of 5 mL/kg. The concentration of VX–809 in plasma samples is determined with a liquid chromatography/tandem MS method. Pharmacokinetic parameters are calculated byusing WinNonlin Professional Edition software, version 4.0.1..References:[1]. Van Goor F, et al. Correction of the F508del–CFTR protein processing defect in vitro by the investigational drug VX–809. Proc Natl Acad Sci U S A. 2011 Nov 15;108(46):18843–8.[2]. Phuan PW, et al. Synergy–based small–molecule screen using a human lung epithelial cell line yields ΔF508–CFTR correctors that augment VX–809 maximal efficacy. Mol Pharmacol. 2014 Jul;86(1):42–51.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

安捷伦产品目录

15
Real-Time PCR
16
Mx3000P QPCR System
17
Brilliant III Ultra-Fast SYBR Green QPCR and QRT-PCR Reagents
18
Brilliant III Ultra-Fast QPCR and QRT-PCR Reagents
Agilent / STRATAGENE
Agilent website: /genomics
Welgene | Agilent Stratagene
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Table of Content
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/ XL1-Red Competent Cells SoloPack Gold Supercompetent Cells
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適用於 UNG 去汙染或 bisulphite
sequencing
適用於 TA Cloning
最高敏感性
取代傳統 Taq 的好選擇
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2
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PCR Enzyme & Instrument
Agilent SureCycler 8800
市場上領先的 cycling 速度和 sample 體積 10 ～ 100 μL 簡易快速可以選擇 96 well 和 384 well 操作盤優秀的溫控設備讓各個 well 都能保持溫度的穩定七吋的高解析度觸控螢幕讓操作上更為簡便可以透過網路遠端操控儀器及監控儀器 Agilent 專業的技術支援可以幫助您應對各種 PCR 的問題

Luc-Pair

Luc-Pair™Duo-Luciferase HS Assay Kit-高灵敏性双荧光素酶检测试剂盒Cat.No.LF004(100reactions)Cat.No.LF005(300reactions)Cat.No.LF006(1000reactions)使用说明书GeneCopoeia,Inc.广州易锦生物技术有限公司9620Medical Center Drive,#101地址：广州科学城揽月路3号F区F801（510663）Rockville,MD20850电话：4006-020-200、************、************ USA网站：301-762-0888866-360-9531***********************©2016GeneCopoeia,Inc.使用说明书Luc-Pair™Duo-Luciferase HS Assay KitI.产品概述II.产品信息及储存条件III.细胞裂解IV.FLuc和RLuc工作液的配制V.荧光素酶检测流程VI.有限使用许可及质保声明I.产品概述对报告基因表达的转录调控的研究常被应用于生物学研究和药物发现。

荧光素酶在基因表达研究中应用最为广泛，其主要包含以下几个优点：1)在广泛动态范围内具有高灵敏度2)在哺乳动物细胞内无荧光素酶、背景极低3)实验重复性好4)成本低5)操作简单萤火虫和海肾荧光素酶都具有快捷、简便、灵敏度高的检测特点，被公认为是理想的报告基因，因为它们具有完全不同的进化起源、酶学结构和底物要求。

萤光素酶报告基因的测定需要用光度计或多功能微孔板检测仪，且发光强度与荧光素酶的数量成正比。

萤火虫荧光素酶（Photinus pyralis）已被证实是检测启动子活性和监测基因转录后调控状态的理想的报告基因。

它是在细胞质中作用的酶，分子量为61kDa并催化下列反应：海肾（Renilla reniformis）荧光素酶是一个36kDa单亚基蛋白质，酶活性不需要翻译后修饰，因此它可以作为一个实时转录报告基因，催化下面的生物发光反应：此体系可以在目的基因的附近监控顺式作用元件的转录激活。

AZ876_SDS_MedChemExpress

Inhibitors, Agonists, Screening LibrariesSafety Data Sheet Revision Date:May-24-2017Print Date:May-24-20171. PRODUCT AND COMPANY IDENTIFICATION1.1 Product identifierProduct name :AZ876Catalog No. :HY-18282CAS No. :898800-26-51.2 Relevant identified uses of the substance or mixture and uses advised againstIdentified uses :Laboratory chemicals, manufacture of substances.1.3 Details of the supplier of the safety data sheetCompany:MedChemExpress USATel:609-228-6898Fax:609-228-5909E-mail:sales@1.4 Emergency telephone numberEmergency Phone #:609-228-68982. HAZARDS IDENTIFICATION2.1 Classification of the substance or mixtureNot a hazardous substance or mixture.2.2 GHS Label elements, including precautionary statementsNot a hazardous substance or mixture.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:AZ⁻876; AZ 876Formula:C24H29N3O3SMolecular Weight:439.57CAS No. :898800-26-54. FIRST AID MEASURES4.1 Description of first aid measuresEye contactRemove any contact lenses, locate eye-wash station, and flush eyes immediately with large amounts of water. Separate eyelids with fingers to ensure adequate flushing. Promptly call a physician.Skin contactRinse skin thoroughly with large amounts of water. Remove contaminated clothing and shoes and call a physician.InhalationImmediately relocate self or casualty to fresh air. If breathing is difficult, give cardiopulmonary resuscitation (CPR). Avoid mouth-to-mouth resuscitation.IngestionWash out mouth with water; Do NOT induce vomiting; call a physician.4.2 Most important symptoms and effects, both acute and delayedThe most important known symptoms and effects are described in the labelling (see section 2.2).4.3 Indication of any immediate medical attention and special treatment neededTreat symptomatically.5. FIRE FIGHTING MEASURES5.1 Extinguishing mediaSuitable extinguishing mediaUse water spray, dry chemical, foam, and carbon dioxide fire extinguisher.5.2 Special hazards arising from the substance or mixtureDuring combustion, may emit irritant fumes.5.3 Advice for firefightersWear self-contained breathing apparatus and protective clothing.6. ACCIDENTAL RELEASE MEASURES6.1 Personal precautions, protective equipment and emergency proceduresUse full personal protective equipment. Avoid breathing vapors, mist, dust or gas. Ensure adequate ventilation. Evacuate personnel to safe areas.Refer to protective measures listed in sections 8.6.2 Environmental precautionsTry to prevent further leakage or spillage. Keep the product away from drains or water courses.6.3 Methods and materials for containment and cleaning upAbsorb solutions with finely-powdered liquid-binding material (diatomite, universal binders); Decontaminate surfaces and equipment by scrubbing with alcohol; Dispose of contaminated material according to Section 13.7. HANDLING AND STORAGE7.1 Precautions for safe handlingAvoid inhalation, contact with eyes and skin. Avoid dust and aerosol formation. Use only in areas with appropriate exhaust ventilation.7.2 Conditions for safe storage, including any incompatibilitiesKeep container tightly sealed in cool, well-ventilated area. Keep away from direct sunlight and sources of ignition.Recommended storage temperature:Powder-20°C 3 years4°C 2 yearsIn solvent-80°C 6 months-20°C 1 monthShipping at room temperature if less than 2 weeks.7.3 Specific end use(s)No data available.8. EXPOSURE CONTROLS/PERSONAL PROTECTION8.1 Control parametersComponents with workplace control parametersThis product contains no substances with occupational exposure limit values.8.2 Exposure controlsEngineering controlsEnsure adequate ventilation. Provide accessible safety shower and eye wash station.Personal protective equipmentEye protection Safety goggles with side-shields.Hand protection Protective gloves.Skin and body protection Impervious clothing.Respiratory protection Suitable respirator.Environmental exposure controls Keep the product away from drains, water courses or the soil. Cleanspillages in a safe way as soon as possible.9. PHYSICAL AND CHEMICAL PROPERTIES9.1 Information on basic physical and chemical propertiesAppearance Off-white to orange (Solid)Odor No data availableOdor threshold No data availablepH No data availableMelting/freezing point No data availableBoiling point/range No data availableFlash point No data availableEvaporation rate No data availableFlammability (solid, gas)No data availableUpper/lower flammability or explosive limits No data availableVapor pressure No data availableVapor density No data availableRelative density No data availableWater Solubility No data availablePartition coefficient No data availableAuto-ignition temperature No data availableDecomposition temperature No data availableViscosity No data availableExplosive properties No data availableOxidizing properties No data available9.2 Other safety informationNo data available.10. STABILITY AND REACTIVITY10.1 ReactivityNo data available.10.2 Chemical stabilityStable under recommended storage conditions.10.3 Possibility of hazardous reactionsNo data available.10.4 Conditions to avoidNo data available.10.5 Incompatible materialsStrong acids/alkalis, strong oxidising/reducing agents.10.6 Hazardous decomposition productsUnder fire conditions, may decompose and emit toxic fumes.Other decomposition products - no data available.11.TOXICOLOGICAL INFORMATION11.1 Information on toxicological effectsAcute toxicityClassified based on available data. For more details, see section 2Skin corrosion/irritationClassified based on available data. For more details, see section 2Serious eye damage/irritationClassified based on available data. For more details, see section 2Respiratory or skin sensitizationClassified based on available data. For more details, see section 2Germ cell mutagenicityClassified based on available data. For more details, see section 2CarcinogenicityIARC: No component of this product present at a level equal to or greater than 0.1% is identified as probable, possible or confirmed human carcinogen by IARC.ACGIH: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by ACGIH.NTP: No component of this product present at a level equal to or greater than 0.1% is identified as a anticipated or confirmed carcinogen by NTP.OSHA: No component of this product present at a level equal to or greater than 0.1% is identified as a potential or confirmed carcinogen by OSHA.Reproductive toxicityClassified based on available data. For more details, see section 2Specific target organ toxicity - single exposureClassified based on available data. For more details, see section 2Specific target organ toxicity - repeated exposureClassified based on available data. For more details, see section 2Aspiration hazardClassified based on available data. For more details, see section 212. ECOLOGICAL INFORMATION12.1 ToxicityNo data available.12.2 Persistence and degradabilityNo data available.12.3 Bioaccumlative potentialNo data available.12.4 Mobility in soilNo data available.12.5 Results of PBT and vPvB assessmentPBT/vPvB assessment unavailable as chemical safety assessment not required or not conducted.12.6 Other adverse effectsNo data available.13. DISPOSAL CONSIDERATIONS13.1 Waste treatment methodsProductDispose substance in accordance with prevailing country, federal, state and local regulations.Contaminated packagingConduct recycling or disposal in accordance with prevailing country, federal, state and local regulations.14. TRANSPORT INFORMATIONDOT (US)This substance is considered to be non-hazardous for transport.IMDGThis substance is considered to be non-hazardous for transport.IATAThis substance is considered to be non-hazardous for transport.15. REGULATORY INFORMATIONSARA 302 Components:No chemicals in this material are subject to the reporting requirements of SARA Title III, Section 302.SARA 313 Components:This material does not contain any chemical components with known CAS numbers that exceed the threshold (De Minimis) reporting levels established by SARA Title III, Section 313.SARA 311/312 Hazards:No SARA Hazards.Massachusetts Right To Know Components:No components are subject to the Massachusetts Right to Know Act.Pennsylvania Right To Know Components:No components are subject to the Pennsylvania Right to Know Act.New Jersey Right To Know Components:No components are subject to the New Jersey Right to Know Act.California Prop. 65 Components:This product does not contain any chemicals known to State of California to cause cancer, birth defects, or anyother reproductive harm.16. OTHER INFORMATIONCopyright 2017 MedChemExpress. The above information is correct to the best of our present knowledge but does not purport to be all inclusive and should be used only as a guide. The product is for research use only and for experienced personnel. It must only be handled by suitably qualified experienced scientists in appropriately equipped and authorized facilities. The burden of safe use of this material rests entirely with the user. MedChemExpress disclaims all liability for any damage resulting from handling or from contact with this product.Caution: Product has not been fully validated for medical applications. For research use only.Tel: 609-228-6898 Fax: 609-228-5909 E-mail: tech@Address: 1 Deer Park Dr, Suite Q, Monmouth Junction, NJ 08852, USA。

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.。

Incucyte

Product Information Presentation, Storage and StabilityThe Incucyte® Fabfluor-pH Antibody Labeling Reagents for antibody internalization are supplied as lyophilized solids in sufficient quantity to label 50 μg of test antibody, when used at the suggested molar ratio (1:3 of test antibody to labeling Fab). The lyophilized solid can be stored at 2-8° C for one year. Once re-hydrated, any unused reagent should be aliquoted and stored at -80° C for up to one year. Avoid repeated freeze-thaw cycles.Incucyte® Fabfluor-pH Antibody Labeling ReagentsFor Antibody Internalization AssaysAntibody Labeling Reagent Rehydrated: -80° C *Excitation and Emission maxima were determined at a pH of 4.5.Fabfluor_quick_guideBackgroundIncucyte ® Fabfluor-pH Antibody Labeling Reagents are designed for quick, easy labeling of Fc-containing test antibodies with a Fab fragment-conjugated pH-sensitive fluorophore. The pH-sensitive dye based system exploits the acidic environment of the lysosomes to quantify in-ternalization of the labeled antibody. As Fabfluor labeled antibodies reside in the neutral extracellular solution (pH 7.4), they interact with cell surface specific antigens and are internalized. Once in the lysosomes, they enter an acidic environment (pH 4.5–5.5) and a substantial in-crease in fluorescence is observed. In the absence of ex-pression of the specific antigen, no internalization occurs and the fluorescence intensity of the labeled antibodies remains low. With the Incucyte ® integrated analysis soft-ware, background fluorescence is minimized. These reagents have been validated for use with a number of different antibodies in a range of cell types. The Incucyte ® Live-Cell Analysis System enables real-time, kinetic eval -uation of antibody internalization.Recommended UseWe recommend that the Incucyte ® Fabfluor-pH Antibody Labeling Reagents are prepared at a stock concentration of 0.5 mg/mL by the addition of 100 μL of sterile water and triturated (centrifuge if solution not clear). The reagent may then be diluted directly into the labeling mixture with test antibody. Do NOT sonicate the solution.Additional InformationThe Fab antibody was purified from antisera by a combination of papain digestion and immunoaffinity chromatography using antigens coupled to agarose beads. Fc fragments and whole IgG molecules have been removed.Human Red (Cat. No. 4722) or Human Orange (Cat. No. 4812)—Based on immunoelectrophoresis and/ or ELISA, the antibody reacts with the Fc portion of human IgG heavy chain but not the Fab portion of human IgG. No antibody was detected against human IgM, IgA or against non-immunoglobulin serum proteins. The anti-body may cross-react with other immunoglobulins from other species.Mouse IgG1 (Cat. No. 4723), IgG2a (Cat. No. 4750) or IgG2b (Cat. No. 4751)—Based on antigen-binding assay and/or ELISA, the antibody reacts with the Fc portion of mouse IgG, IgG2a or IgG2b, respectively, but not the Fab portion of mouse immunoglobulins. No antibody was detected against mouse IgM or against non–immunoglobulin serum proteins. The antibody may cross-react with other mouse IgG subclasses or with immunoglobulins from other species.Rat (Cat. No. 4737)—Based on immunoelectrophoresis and/or ELISA, the antibody reacts with the Fc portion of rat IgG heavy chain but not the Fab portion of rat IgG. No antibody was detected against rat IgM, IgA or against non-immunoglobulin serum proteins. The antibody may cross-react with other immunoglobulins from other species.A.B.C.D.R e d O b j e c t A r e a (x 105 μm 2 p e r w e l l )Time (hours)A U C x 106 (0–12 h )log [α–CD71] (g/mL)Example DataFigure 1: Concentration-dependent increase in antibody internalization of Incucyte ® Fabfluor labeled-α-CD71 in HT1080 cells. α-CD71 and mouse IgG1 isotype control were labeled with Incucyte ® Mouse IgG1 Fabfluor-pH Red Antibody Labeling Reagent. HT1080 cells were treated with either Fabfluor-α-CD71 or Fabfluor-IgG1 (4 μg/mL); HD phase and red fluorescence images were captured every 30 minutes over 12 hours using a 10X magnification. (A) Images of cells treated with Fabfluor-α-CD71 display red fluorescence in the cytoplasm (images shown at 6 h). (B) Cells treated with labeled isotype control display no cellular fluorescence. (C) Time-course of Fabfluor-α-CD71 internalization with increasing concentrations of Fabfluor-α-CD71 (progressively darker symbols). Internalization has been quantified as the red object area for each time-point. (D) Concentration response curve to Fabfluor-α-CD71. Area under the curve (AUC) values have been determined from the time-course shown in panel C (0-12 hours) and are presented as the mean ± SEM, n=3 wells.CD71-FabfluorIgG-FabfluorProtocols and ProceduresMaterialsIncucyte® Fabfluor-pH Antibody Labeling ReagentTest antibody of interest containing human, mouse, or rat IgG Fc region (at known concentration)Target cells of interestTarget cell growth mediaSterile distilled water96-well flat bottom microplate (e.g. Corning Cat. No. 3595) for imaging96-well round black round bottom ULA plate (e.g. Corning Cat. No. 45913799) or amber microtube (e.g. Cole Parmer Cat. No. MCT-150-X, autoclaved) for conjugation step0.01% Poly-L-Ornithine (PLO) solution (e.g. Sigma Cat. No. P4957), optional for non-adherent cells Recommended control antibodiesIt is strongly recommended that a positive and negative control is run alongside test antibodies and cell lines. For example, CD71, which is a mouse anti-human antibody, is recommended as a positive control for the mouse Fab.Anti-CD71, clone MEM-189, IgG1 e.g. Sigma Cat. No. SAB4700520-100UGAnti-CD71, clone CYG4, IgG2a e.g. BioLegend Cat. No. 334102Isotype controls, depending on isotype being studied—Mouse IgG1, e.g. BioLegend Cat. No. 400124, Mouse IgG2a e.g. BioLegend Cat. No. 401501Preparation of Incucyte® Antibody Internalization Assay 1. Seed target cells of interest1.1 Harvest cells of interest and determine cell concentra-tion (e.g. trypan blue + hemocytometer).1.2 Prepare cell seeding stock in target cell growth mediawith a cell density to achieve 40–50% confluence be-fore the addition of labeled antibodies. The suggested starting range is 5,000–30,000 cells/well, although the seeding density will need to be optimized for each cell type.Note: For non-adherent cell types, a well coating may be required to maintain even cell distribution in the well. For a 96-well flat bottom plate, we recommend coating with 50 μL of either 0.01% Poly-L-Or-nithine (PLO) solution or 5 μg/mL fibronectin diluted in 0.1% BSA.Coat plates for 1 hour at ambient temperature, remove solution from wells and then allow the plates to dry for 30-60 minutes prior to cell addition.1.3 Using a multi-channel pipette, seed cells (50 µL perwell) into a 96-well flat bottom microplate. Lightly tapplate side to ensure even liquid distribution in well. Toensure uniform distribution of cells in each well, allowthe covered plate sit on a level surface undisturbed at room temperature in the tissue culture hood for 30minutes. After cells are settled, place the plate insidethe Incucyte® Live-Cell Analysis System to monitor cell confluence.Note: Depending on cell type, plates can be used in assay once cells have adhered to plastic and achieved normal cell morphology e.g.2-3 hours for HT1080 or 1-2 hours for non-adherent cell types. Some cell types may require overnight incubation.2. Label Test Antibody2.1 Rehydrate the Incucyte® Fabfluor-pH Antibody Label-ing Reagent with 100 µL sterile water to result in a final concentration of 0.5 mg/mL. Triturate to mix (centrifuge if solution is not clear).Note: The reagent is light sensitive and should be protected fromlight. Rehydrated reagent can be aliquoted into amber or foilwrapped tubes and stored at -80° C for up to 1 year (avoid freezing and thawing).2.2 Mix test antibody with rehydrated Incucyte® Fabfluor–pH Antibody Labeling Reagent and target cell growth media in a black round bottom microplate or ambertube to protect from light (50 µL/well).a. Add test antibody and Incucyte® Fabfluor–pH Anti-body Labeling Reagent at 2X the final concentration.We suggest optimizing the assay by starting with afinal concentration of 4 µg/mL of test antibody or theFabfluor-pH Antibody Labeling Reagent (i.e. 2Xworking concentration = 8 µg/mL).Note: A 1:3 molar ratio of test antibody to Incucyte® Fabfluor-pHAntibody Labeling Reagent is recommended. The labeling re-agent is a third of the size of a standard antibody (50 and 150KDa, respectively). Therefore, labeling equal quantities will pro-duce a 1:3 molar ratio of test antibody to labeling Fab.b. Make sufficient volume of 2X labeling solution for50 µL/well for each sample. Triturate to mix.c. Incubate at 37° C for 15 minutes protected from light.Note: If performing a range of concentrations of test antibody,e.g. concentration response-curve, it is recommended to createthe dilution series post the conjugation step to ensure consistentmolar ratio. We strongly recommend the use of both a negativeand positive control antibody in the same plate.3. Add labeled antibody to cells3.1 Remove cell plate from incubator.3.2 Using a multi-channel pipette, add 50 µL of 2X labeledantibody and control solutions to designated wells.Remove any bubbles and immediately place plate in the Incucyte® Live-Cell Analysis System and start scanning.Note: To reduce the risk of condensation formation on the lid priorto first image acquisition, maintain all reagents at 37° C prior toplate addition.4. Acquire images and analyze4.1 In the Incucyte® Software, schedule to image every15-30 minutes, depending on the speed of the specific antibody internalization.a Scan on schedule, standard. If the Incucyte® Cell-by-Cell Analysis Software Module (Cat. No. 9600-0031)is available, adherent cell-by-cell or non-adherentcell-by-cell scan types can be selected.b Channel selection: select “phase” and “red” or“phase” and "orange” (depending on reagent used).c Objective: 10X or 20X depending on cell types used,generally 10X is recommended for adherent cells,and 20X for non-adherent or smaller cells.NOTE: The optional Incucyte® Cell-by-Cell Analysis SoftwareModule enables the classification of cells into sub-populationsbased on properties including fluorescence intensity, size andshape. For further details on this analysis module and its appli-cation, please see: /cell-by-cell.4.2 To generate the metrics, user must create an AnalysisDefinition suited to the cell type, assay conditions andmagnification selected.4.3 Select images from a well containing a positiveinternalization signal and an isotype control well(negative signal) at a time point where internalizationis visible.4.4 In the Analysis Definition:Basic Analyzer:a. Set up the mask for the phase confluence measurewith fluorescence channel turned off.b. Once the phase mask is determined, turn the fluores-cence channel on: Exclude background fluorescencefrom the mask using the background subtractionfeature. The feature “Top-Hat” will subtract localbackground from brightly fluorescent objects withina given radius; this is a useful tool for analyzing ob-jects which change in fluorescence intensity overtime.i The radius chosen should reflect the size of thefluorescent object but contain enough backgroundto reliably estimate background fluorescence inthe image; 20-30 μm is often a useful startingpoint.ii The threshold chosen will ensure that objectsbelow a fluorescence threshold will not bemasked.iii Choose a threshold in which red or orange objectsare masked in the positive response image but lownumbers in the isotype control, negative responsewell. For a very sensitive measurement, for example,if interested in early responses, we suggest athreshold of 0.2.NOTE: The Adaptive feature can be used for analysis but maynot be as sensitive and may miss early responses. If interestedin rate of response, Top-Hat may be preferable.Cell-by-Cell (if available):a. Create a Cell-by-Cell mask following the softwaremanual.b. There is no need to separate phase and fluorescencemasks. The default setting of Top-Hat No Mask forthe fluorescence channel will enable backgroundsubtraction without generation of a mask. Ensurethat the Top-Hat radius is set to a value higher thanthe radius of the larger clusters to avoid excess back-ground subtraction.c. The threshold of fluorescence can be determined inCell-by-Cell Classification.Specifications subject to change without notice.© 2020. All rights reserved. Incucyte, Essen BioScience, and all names of Essen BioScience prod -ucts are registered trademarks and the property of Essen BioScience unless otherwise specified. Essen BioScience is a Sartorius Company. Publication No.: 8000-0728-A00Version 1 | 2020 | 04Sales and Service ContactsFor further contacts, visit Essen BioScience, A Sartorius Company /incucyte Sartorius Lab Instruments GmbH & Co. KGOtto-Brenner-Strasse 20 37079 Goettingen, Germany Phone +49 551 308 0North AmericaEssen BioScience Inc. 300 West Morgan Road Ann Arbor, Michigan, 48108USATelephone +1 734 769 1600E-Mail:***************************EuropeEssen BioScience Ltd.Units 2 & 3 The Quadrant Newark CloseRoyston Hertfordshire SG8 5HLUnited KingdomTelephone +44 (0) 1763 227400E-Mail:***************************APACEssen BioScience K.K.4th floor Daiwa Shinagawa North Bldg.1-8-11 Kita-Shinagawa Shinagawa-ku, Tokyo 140-0001 JapanTelephone: +81 3 6478 5202E-Mail:*************************5. Analysis GuidelinesAs the labeled antibody is internalized into the acidic environment of the lysosome, the area of fluorescence intensity inside the cells increases.This can be reported in two ways:Ways to Report Basic AnalyzerCell-by-Cell Analysis* To correct for cell proliferation, it is advisable to normalize the fluorescence area to the total cell area using User Defined Metrics.For Research Use Only. Not For Therapeutic or Diagnostic Use.LicensesFor non-commercial research use only. Not for therapeutic or in vivo applications. Other license needs contact Essen BioS cience.Fabfluor-pH Red Antibody Labeling Reagent: This product or portions thereof is manufactured under license from Carnegie Mellon University and U.S. patent numbers 7615646 and 8044203 and related patents. This product is licensed for sale only for research. It is not licensed for any other use. There is no implied license hereunder for any commercial use.Fabfluor-pH Orange Antibody Labeling Reagent: This product or portions thereof is manufactured under a license from Tokyo University and is covered by issued patents EP2098529B1, JP5636080B2, US8258171, and US9784732 and related patent applications. This product and related products are trademarks of Goryo Chemical. Any application of above mentioned technology for commercial purpose requires a separate li -cense from: Goryo Chemical, EAREE Bldg., SF Kita 8 Nishi 18-35-100, Chuo-Ku, Sapporo, 060-0008 Japan.SupportA complete suite of cell health applications is available to fit your experimental needs. Find more information at /incucyte Foradditionalproductortechnicalinformation,************************************************************/incucyte。

红色REDTaq DNA多合物说明书

REDTaq® DNA PolymeraseCatalog Number D4309Storage Temperature –20 ︒CTECHNICAL BULLETINProduct DescriptionREDTaq DNA Polymerase is a unique blend of our high quality Taq DNA Polymerase combined with an inert red dye. The dye enables quick visual recognition of reactions to which enzyme has been added, as well as confirmation of complete mixing. The formulation allows aliquots (5-10 μL) from the PCR to be directly loaded onto an agarose gel without addition of electrophoresis loading buffers. The red dye migrates at the same rate as a 125 bp fragment in a 1% agarose gel. Because gel loading buffer is not added to the reaction mix, a sample can be re-amplified, such as in nested PCR.The red dye has no effect on automated or manual DNA sequencing, ligase mediated ligations, or exonucleolytic PCR product digestion. Though exceptions may exist, the dye is generally inert in restriction enzyme digestions. If desired, the dye can be removed from the amplicon using any standard purification method.The enzyme is provided at one unit/μL for more accurate volume measurement and less waste. Reactions using REDTaq DNA Polymerase and its optimized 10⨯ PCR buffer are formulated as any PCR mixtures. There are no additional reaction preparation steps or protocol changes required.Unit Definition: One unit incorporates 10 nmol of total deoxyribonucleoside-triphosphates into acid precipitable DNA in 30 min at 74 ︒C.Reagents Provided∙REDTaq DNA Polymerase, Catalog Number D56841 unit/μL in 20 mM Tris-HCl, pH 8.0, 100 mM KCl,0.1 mM EDTA, 1 mM DTT, stabilizers, inert dye,50% glycerol. Provided as 50, 250, 1,000 or 2,500 units∙ 10⨯ REDTaq PCR Reaction Buffer, Catalog Number B5926, 100 mM Tris-HCl, pH 8.3, 500mM KCl, 11 mM MgCl2 and 0.1% gelatin. Provided as 1 ml package Reagents and equipment required but not provided∙Deoxynucleotide Mix, Catalog Number D729510 mM each dATP, dCTP, dGTP, and TTP∙Water, PCR Reagent, Catalog Number W1754∙Mineral Oil, Catalog Number M8662 (optional)∙ Primers∙DNA to be amplified∙ Dedicated pipettes∙PCR pipette tips∙0.5 ml or 0.2 ml thin-walled PCR tubes, Catalog Numbers P3114 and P3364∙ Thermal cyclerPrecautions and DisclaimerThis product is for R&D use only, not for drug, household, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices.StorageStore all components at –20 ︒C.ProcedureNote: REDTaq 10⨯ PCR Reaction Buffer has been formulated to be compatible with REDTaq DNA Polymerase. If other buffers are to be used, they must be formulated to account for 0.4 mM magnesium being added to the PCR from the enzyme. In this case the final enzyme concentration in the PCR is assumed to be 0.05 units/μL Other enzyme concentrations may require further magnesium concentration optimization. Optimal concentrations of template DNA, MgCl2, KCl, and PCR adjuncts, as well as pH, are often target specific. It may be necessary to determine the optimal concentration of each component.21. Add the following reagents to a thin-walled 200 μLor 500 μL PCR tube in the order listed below. Amount Component FinalConcentration5 μL 10⨯ REDTaq PCRReaction Buffer1⨯1 μL Deoxynucleotide Mix,D7295 200 μM (each dNTP)- μL Primer 0.1-0.5 μM- μL Primer 0.1-0.5 μM2.5 μL REDTaq DNAPolymerase0.05 unit/μL- μL Template DNA(typically 10 ng)200 pg/μL q.s. Water (Cat. # W1754)50 μL Total volumeNote 1: A master mix is highly recommended when setting up multiple reactions.Note 2:10x REDTaq PCR Reaction Buffer should be vortexed vigorously prior to use.2. Mix gently by vortex and briefly centrifuge tocollect all components to the bottom of the tube.3. Add 50 μL of mineral oil to the top of each tube toprevent evaporation if not using a thermal cyclerwith a heated lid.4. Optimum cycling parameters vary with PCRcomposition (i.e. primer sequences, template,MgCl2 concentration etc.) and thermal cycler. Itmay be necessary to optimize the cyclingparameters to achieve maximum product yieldand/or quality. Typical cycling parameters are:.For cycles 1-30Denaturation 94 ︒C 1 min Annealing 55 ︒C 2 min Extension 72 ︒C 3 min Note: 25-30 cycles of amplification are recommended.5. The amplified DNA can be evaluated by agarosegel electrophoresis by loading 5-10 μL of the PCR reaction onto the gel without the addition of gelloading buffers. Note: A minimum of 1.5 units of REDTaq DNA polymerase must be added per 50 μL reaction for unencumbered gel loading. The red dye migrates as a 125 bp fragment in a 1% agarose gel.If a more intense tracking dye is desired, an unused lane can be used to run any common tracking dye (i.e. as provided by a DNA marker). Alternatively, a tracking solution devoid of DNA can be added to a previously loaded REDTaq PCR product well. Amplification products can be visualized by standard methodologies (e.g. ethidium bromide staining). Mineral oil overlay may be removed by a single chloroform extraction (1:1), recovering the aqueous phase. Alternatively, an aliquot of the aqueous phase can be taken by withdrawing solution from below the aqueousphase/mineral oil interface.Related ProductsReagents and Kits∙Lambda Hind III DNA Marker, Catalog Number D9780∙Enhanced Avian HS RT-PCR kits, Catalog Number HSRT100 (100 reactions). The RT-PCR kit combines twopowerful techniques to convert mRNA into cDNA andsubsequently to amplify the cDNA. The enhanced avianreverse transcriptase has an enhanced ability to transcribe through difficult secondary structure at elevatedtemperatures (up to 65 ︒C). Includes JumpStart AccuTaq™LA for hot-start PCR.Equipment∙PCR Multiwell Plate, 96-well, Catalog No. Z374903∙PCR Multiwell Plate, 384-well, Catalog No. Z374911∙PCR Microtubes, 0.2 ml, attached caps, Catalog No.Z374873∙PCR Microtubes, 0.2 ml strip tubes with strip caps, Catalog No. Z374962∙Sealing accessory for PCR vessels, Micro Mats, Catalog No. Z374938∙PCR Workstation, 120V, Catalog No. Z376213∙PCR Workstation, 240V, Catalog No. Z376221NOTICE TO PURCHASER: LIMITED LICENSEUse of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: US 8,404,464 and US 7,972,828. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims.REDTaq is a registered trademark of Sigma-Aldrich Co, LLCGAR,PHC,KNV 10/16-1©2016 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at and/or on the reverse side of the invoice or packing slip.。

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Product Name:
VX-809CAS No.:
936727-05-8
Product Data Sheet
Cat. No.:
HY-13262MWt:
452.41Formula:
C24H18F2N2O5Purity :
>98%
Solubility:Mechanisms:
Biological Activity:
Pathways:Membrane Transporter/Ion Channel; Target:CFTR DMSO ≥90mg/mL Water
<1.2mg/mL Ethanol ≥
4.6mg/mL
g y VX-809 is the second investigational oral candidate compound for the treatment of cystic fibrosis (CF). VX-809 may act to restore the function of the cystic fibrosis transmembrane conductance
regulator (CFTR) protein, the defective cell membrane protein responsible for the progression of CF.VX-809 defects in the CFTR protein affect the transport of chloride and other ions across cells, and lead to the accumulation of thick, sticky mucus in the lungs of patients with CF. This mucus fosters chronic infection and inflammation, and results in irreversible lung damage.
References:
[1]. Clancy JP et al. Results of a phase IIa study of VX-809, an investigational CFTR corrector compound, in subjects with cystic fibrosis homozygous for the F508del-CFTR mutation. Thorax
doi:10.1136[2]. Pettit RS.Cystic fibrosis transmembrane conductance regulator-modifying medications: the []y g y g
future of cystic fibrosis treatment.Ann Pharmacother. 2012 Jul;46(7-8):1065-75. Epub 2012 Jun 26.[3]. Lubamba B, Dhooghe B, Noel S, Leal T.Cystic fibrosis: Insight into CFTR pathophysiology and
pharmacotherapy.Clin Biochem. 2012 Jun 12.[4]. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Stack JH, Straley KS, Decker CJ, Miller M,McCartney J, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu PA.Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.Proc Natl Acad Sci U S
A. 2011 Nov 15;108(46):18843-8. Epub 2011 Oct ...
Caution: Not fully tested. For research purposes only
Medchemexpress LLC
18 W i l k i n s o n W a y , P r i n c e t o n , N J 08540,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。