TAK-779_229005-80-5_DataSheet_MedChemExpress
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 ]. 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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。
GOTS4.0实施手册中英文版(北京爱科赛尔认证中心有限公司)
Copyright: © 2014 by 'Global Organic Textile Standard International Working Group' © 2014 版权所有人：全球有机纺织品标准国际工作组（IWG）
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GOTS Manual- Issue of 01 March 2014, page 1 of 43 GOTS 手册-2014 年 3 月 1 日发布，第 1 页，共 43 页
Table of contents
目录 Principles........................................................................................................................... 4 总则 ................................................................................................................................... 4 Official interpretations for specific criteria of the GOTS, Version 4.0 .................................. 5 4.0 版 GOTS 特定准则的官方释义 ..................................................................................... 5 1.2 Scope and structure................................
ICP-MS法测定富马酸福莫特罗原料药中催化剂钯的残留量
IC P-M S法测定富马酸福莫特罗原料药中催化剂钯的残留量李春盈，张静，钟依雯，曾敏珊（广州市药品检验所，广东广州510160)摘要：目的建立富马酸福莫特罗中催化剂钯（P d)残留的检测方法。
方法通过直接溶解样品，以电感耦合等离子质谱法(1CP-MS)测定样品中钯的含量。
结果测定了3批富马酸福莫特罗中钯的含量。
ICP-MS条件经优化后，测得钯在2~50 浓度范围内相关系数为0.999 9(^1 = 6)，线性关系良好，方法检测限为0.03 fxg.L_1,回收率在98.0%〜101.4%之间，ftSZ)为l.l%(n = 6)。
结论本方法操作简便、快捷、灵敏度高，适用于富马酸福莫特罗中钯残留量的检测。
关键词：电感耦合等离子质谱法;钯残留量；富马酸福莫特罗中图分类号:R917 文献标志码:A文章编号：1674-229X( 2020) 11-0767-02Doi ： 10.12048/j.issn. 1674-229X.2020.11.009Determination of Catalyst Palladium Residue in Formoterol Fumarate Dihydrate Crude Product by ICP-MS LI Chunying, ZHANG Jing,ZHONG \iwen,ZENG Minshan( Giia/igz/low Institute of Drug Control, Guangzhou ^ Guangdong 5\0\60 y China)ABSTRACT ： OBJECTIVE To establish a method for determinating the residual content of palladium which was used as a catalyst in drug substance of formoterol fumarate dihydrate. METHODS The samples were prepared through dissolving directly and analyzed by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS The content of palladium residue in 3 batches of formoterol fumarate dihydratewere determined.Optimized by ICP-MS, the calibration curves of palladium were at good linear of the concentration range of 2-50 fxg • L 1 , with correlation coefficient (r) was 0.999 9 (n = 6).Detection limits of the method was 0.03 ixg'L-1. The recoveries were in the range of 98.0%-101.4%,and RSD was 1.1% (n = 6).CONCLUSION The method is highly sensitive, rapid and accuracy for determination of catalyst palladium residue in formoterol fumarate dihydrate crude product.KEY WORDS：ICP-MS；palladium residue；formoterol fumarate dihydrate富马酸福莫特罗最初是由日本山之内制药公司 开发的长效、高选择性p2受体激动剂，用于治疗支 气管哮喘，急性支气管炎或喘息性支气管炎等气道 阻塞性肺病[M]。
稳定性英文版
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFLUOXETINE HClC17H18F3NO•HClM.W. = 345.79CAS — 59333-67-4STABILITY INDICATINGA S S A Y V A L I D A T I O NMethod is suitable for:ýIn-process controlþProduct ReleaseþStability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANY MECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationTABLE OF CONTENTS INTRODUCTION........................................................................................................................ PRECISION............................................................................................................................... System Repeatability ................................................................................................................ Method Repeatability................................................................................................................. Intermediate Precision .............................................................................................................. LINEARITY................................................................................................................................ RANGE...................................................................................................................................... ACCURACY............................................................................................................................... Accuracy of Standard Injections................................................................................................ Accuracy of the Drug Product.................................................................................................... VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION........................................... Linearity at Low Concentrations................................................................................................. Accuracy of Fluoxetine HCl at Low Concentration..................................................................... System Repeatability................................................................................................................. Quantitation Limit....................................................................................................................... Detection Limit........................................................................................................................... VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES).................................. Meta-Fluoxetine HCl linearity at 0.05% - 1.0%........................................................................... Detection Limit for Fluoxetine HCl.............................................................................................. Quantitation Limit for Meta Fluoxetine HCl................................................................................ Accuracy for Meta-Fluoxetine HCl ............................................................................................ Method Repeatability for Meta-Fluoxetine HCl........................................................................... Intermediate Precision for Meta-Fluoxetine HCl......................................................................... SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD............................. FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD..................................1. Unstressed analysis...............................................................................................................2. Acid Hydrolysis stressed analysis..........................................................................................3. Base hydrolysis stressed analysis.........................................................................................4. Oxidation stressed analysis...................................................................................................5. Sunlight stressed analysis.....................................................................................................6. Heat of solution stressed analysis.........................................................................................7. Heat of powder stressed analysis.......................................................................................... System Suitability stressed analysis.......................................................................................... Placebo...................................................................................................................................... STABILITY OF STANDARD AND SAMPLE SOLUTIONS......................................................... Standard Solution...................................................................................................................... Sample Solutions....................................................................................................................... ROBUSTNESS.......................................................................................................................... Extraction................................................................................................................................... Factorial Design......................................................................................................................... CONCLUSION...........................................................................................................................ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationBACKGROUNDTherapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptake inhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloride has been shown to have comparable efficacy to tricyclic antidepressants but with fewer anticholinergic side effects. The patent expiry becomes effective in 2001 (US). INTRODUCTIONFluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosage strengths with the same capsule weight. The formulas are essentially similar and geometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20 mg strength.The following validation, for the method SI-IAG-206-02 , includes assay and determination of Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06. Currently the method is the in-house method performed for Stability Studies. The Validation was performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.In the forced degradation studies, the two placebo samples were also used. PRECISIONSYSTEM REPEATABILITYFive replicate injections of the standard solution at the concentration of 0.4242mg/mL as described in method SI-IAG-206-02 were made and the relative standard deviation (RSD) of the peak areas was calculated.SAMPLE PEAK AREA#15390#25406#35405#45405#55406Average5402.7SD 6.1% RSD0.1ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::PRECISION - Method RepeatabilityThe full HPLC method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method repeated six times and the relative standard deviation (RSD) was calculated.SAMPLENumber%ASSAYof labeled amountI 96.9II 97.8III 98.2IV 97.4V 97.7VI 98.5(%) Average97.7SD 0.6(%) RSD0.6PRECISION - Intermediate PrecisionThe full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-21-001 for the 20mg dosage form. The method was repeated six times by a second analyst on a different day using a different HPLC instrument. The average assay and the relative standard deviation (RSD) were calculated.SAMPLENumber% ASSAYof labeled amountI 98.3II 96.3III 94.6IV 96.3V 97.8VI 93.3Average (%)96.1SD 2.0RSD (%)2.1The difference between the average results of method repeatability and the intermediate precision is 1.7%.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationLINEARITYStandard solutions were prepared at 50% to 200% of the nominal concentration required by the assay procedure. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over the concentration range required. Y-Intercept was found to be insignificant.RANGEDifferent concentrations of the sample (IAG-21-001) for the 20mg dosage form were prepared, covering between 50% - 200% of the nominal weight of the sample.Conc. (%)Conc. (mg/mL)Peak Area% Assayof labeled amount500.20116235096.7700.27935334099.21000.39734463296.61500.64480757797.52000.79448939497.9(%) Average97.6SD 1.0(%) RSD 1.0ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::RANGE (cont.)The results demonstrate linearity as well over the specified range.Correlation coefficient (RSQ)0.99981 Slope11808.3Y -Interceptresponse at 100%* 100 (%) 0.3%ACCURACYACCURACY OF STANDARD INJECTIONSFive (5) replicate injections of the working standard solution at concentration of 0.4242mg/mL, as described in method SI-IAG-206-02 were made.INJECTIONNO.PEAK AREA%ACCURACYI 539299.7II 540599.9III 540499.9IV 5406100.0V 5407100.0Average 5402.899.9%SD 6.10.1RSD, (%)0.10.1The percent deviation from the true value wasdetermined from the linear regression lineHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::ACCURACY OF THE DRUG PRODUCTAdmixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl were prepared at the same proportion as in a capsule (70%-180% of the nominal concentration).Three preparations were made for each concentration and the recovery was calculated.Conc.(%)Placebo Wt.(mg)Fluoxetine HCl Wt.(mg)Peak Area%Accuracy Average (%)70%7079.477.843465102.27079.687.873427100.77079.618.013465100.0101.0100%10079.6211.25476397.910080.8011.42491799.610079.6011.42485498.398.6130%13079.7214.90640599.413080.3114.75632899.213081.3314.766402100.399.618079.9920.10863699.318079.3820.45879499.418080.0820.32874899.599.4Placebo, Batch Lot IAG-21-001HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION OF FLUOXETINE HClAT LOW CONCENTRATIONLINEARITY AT LOW CONCENTRATIONSStandard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of the working concentration required by the method SI-IAG-206-02. Linear regression analysis demonstrated acceptability of the method for quantitative analysis over this range.ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATIONThe peak areas of the standard solution at the working concentration were measured and the percent deviation from the true value, as determined from the linear regression was calculated.SAMPLECONC.µg/100mLAREA FOUND%ACCURACYI 470.56258499.7II 470.56359098.1III 470.561585101.3IV 470.561940100.7V 470.56252599.8VI 470.56271599.5(%) AverageSlope = 132.7395299.9SD Y-Intercept = -65.872371.1(%) RSD1.1HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSystem RepeatabilitySix replicate injections of standard solution at 0.02% and 0.05% of working concentration as described in method SI-IAG-206-02 were made and the relative standard deviation was calculated.SAMPLE FLUOXETINE HCl AREA0.02%0.05%I10173623II11503731III10103475IV10623390V10393315VI10953235Average10623462RSD, (%) 5.0 5.4Quantitation Limit - QLThe quantitation limit ( QL) was established by determining the minimum level at which the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02% of the working standard concentration with resulting RSD (for six injections) of 5.0%. Detection Limit - DLThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected. The detection limit of Fluoxetine HCl is about 0.01% of the working standard concentration.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::VALIDATION FOR META-FLUOXETINE HCl(EVALUATING POSSIBLE IMPURITIES)Meta-Fluoxetine HCl linearity at 0.05% - 1.0%Relative Response Factor (F)Relative response factor for Meta-Fluoxetine HCl was determined as slope of Fluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs (analysed at the same time).F =132.7395274.859534= 1.8Detection Limit (DL) for Fluoxetine HClThe detection limit (DL) was established by determining the minimum level at which the analyte was reliably detected.Detection limit for Meta Fluoxetine HCl is about 0.02%.Quantitation Limit (QL) for Meta-Fluoxetine HClThe QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-Fluoxetine HCl can be quantified with acceptable accuracy and precision.Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HCl solution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into the HPLC and the recovery was calculated.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES].Approx.Conc.(%)Known Conc.(µg/100ml)Area in SpikedSampleFound Conc.(µg/100mL)Recovery (%)0.0521.783326125.735118.10.0521.783326825.821118.50.0521.783292021.55799.00.0521.783324125.490117.00.0521.783287220.96996.30.0521.783328526.030119.5(%) AVERAGE111.4SD The recovery result of 6 samples is between 80%-120%.10.7(%) RSDQL for Meta Fluoxetine HCl is 0.05%.9.6Accuracy for Meta Fluoxetine HClDetermination of Accuracy for Meta-Fluoxetine HCl impurity was assessed using triplicate samples (of the drug product) spiked with known quantities of Meta Fluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and 120% of the specified limit - 0.05%).The results are within specifications:For 0.4% and 0.5% recovery of 85% -115%For 0.6% recovery of 90%-110%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::META-FLUOXETINE HCl[RECOVERY IN SPIKED SAMPLES]Approx.Conc.(%)Known Conc.(µg/100mL)Area in spikedSample Found Conc.(µg/100mL)Recovery (%)[0.4%]0.4174.2614283182.66104.820.4174.2614606187.11107.370.4174.2614351183.59105.36[0.5%]0.5217.8317344224.85103.220.5217.8316713216.1599.230.5217.8317341224.81103.20[0.6%]0.6261.3918367238.9591.420.6261.3920606269.81103.220.6261.3920237264.73101.28RECOVERY DATA DETERMINED IN SPIKED SAMPLESHPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::REPEATABILITYMethod Repeatability - Meta Fluoxetine HClThe full method (as described in SI-IAG-206-02) was carried out on the finished drug product representing lot number IAG-21-001-(1). The HPLC method repeated serially, six times and the relative standard deviation (RSD) was calculated.IAG-21-001 20mg CAPSULES - FLUOXETINESample% Meta Fluoxetine % Meta-Fluoxetine 1 in Spiked Solution10.0260.09520.0270.08630.0320.07740.0300.07450.0240.09060.0280.063AVERAGE (%)0.0280.081SD 0.0030.012RSD, (%)10.314.51NOTE :All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta Fluoxetine HCl were injected.HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationED. N0: 04Effective Date:APPROVED::Intermediate Precision - Meta-Fluoxetine HClThe full method as described in SI-IAG-206-02 was applied on the finished product IAG-21-001-(1) .It was repeated six times, with a different analyst on a different day using a different HPLC instrument.The difference between the average results obtained by the method repeatability and the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HCl as is and 28.5% for spiked solution).IAG-21-001 20mg - CAPSULES FLUOXETINESample N o:Percentage Meta-fluoxetine% Meta-fluoxetine 1 in spiked solution10.0260.06920.0270.05730.0120.06140.0210.05850.0360.05560.0270.079(%) AVERAGE0.0250.063SD 0.0080.009(%) RSD31.514.51NOTE:All results obtained were well below the QL (0.05%) thus spiked samples slightly greater than 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solution was 14.5%HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSPECIFICITY - STABILITY INDICATING EVALUATIONDemonstration of the Stability Indicating parameters of the HPLC assay method [SI-IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array detector was incorporated utilizing a commercial chromatography software managing system2, and applied to analyze a range of stressed samples of the finished drug product.GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):Purity Angle-is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in the integrated peak against the peak apex spectrum.Purity Threshold-is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.Match Angle-is a comparison of the spectrum at the peak apex against a library spectrum.Match Threshold-is the sum of the match noise angle3 and match solvent angle4.3Noise Angle-is a measure of spectral non-homogeneity caused by system noise.4Solvent Angle-is a measure of spectral non-homogeneity caused by solvent composition.OVERVIEWT he assay of the main peak in each stressed solution is calculated according to the assay method SI-IAG-206-02, against the Standard Solution, injected on the same day.I f the Purity Angle is smaller than the Purity Threshold and the Match Angle is smaller than the Match Threshold, no significant differences between spectra can be detected. As a result no spectroscopic evidence for co-elution is evident and the peak is considered to be pure.T he stressed condition study indicated that the Fluoxetine peak is free from any appreciable degradation interference under the stressed conditions tested. Observed degradation products peaks were well separated from the main peak.1® PDA-996 Waters™ ; 2[Millennium 2010]ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationFORCED DEGRADATION OF FINISHED PRODUCT & STANDARD 1.UNSTRESSED SAMPLE1.1.Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 98.5%.SAMPLE - UNSTRESSEDFluoxetine:Purity Angle:0.075Match Angle:0.407Purity Threshold:0.142Match Threshold:0.4251.2.Standard solution was prepared as stated in method SI-IAG-206-02 and injected into the HPLC system. The calculated assay is 100.0%.Fluoxetine:Purity Angle:0.078Match Angle:0.379Purity Threshold:0.146Match Threshold:0.4272.ACID HYDROLYSIS2.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system after filtration.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.SAMPLE- ACID HYDROLYSISFluoxetine peak:Purity Angle:0.055Match Angle:0.143Purity Threshold:0.096Match Threshold:0.3712.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc. HCl were added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSTANDARD - ACID HYDROLYSISFluoxetine peak:Purity Angle:0.060Match Angle:0.060Purity Threshold:0.099Match Threshold:0.3713.BASE HYDROLYSIS3.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.SAMPLE - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.063Match Angle:0.065Purity Threshold:0.099Match Threshold:0.3623.2.Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours, then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity did NOT decrease - 99.5%.STANDARD - BASE HYDROLYSISFluoxetine peak:Purity Angle:0.081Match Angle:0.096Purity Threshold:0.103Match Threshold:0.3634.OXIDATION4.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent added and the solution sonicated for 10 minutes.1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then made up to volume with Diluent, filtered and injected into HPLC system.Fluoxetine peak intensity decreased to 95.2%.ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSAMPLE - OXIDATIONFluoxetine peak:Purity Angle:0.090Match Angle:0.400Purity Threshold:0.154Match Threshold:0.4294.2.Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of 30% H2O2 were added to this solution which was standing for 5 hours, made up to volume with Diluent and injected into the HPLC system.Fluoxetine peak intensity decreased to 95.8%.STANDARD - OXIDATIONFluoxetine peak:Purity Angle:0.083Match Angle:0.416Purity Threshold:0.153Match Threshold:0.4295.SUNLIGHT5.1.Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST was set to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%. The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak was observed at RRT of 1.5 (2.7%).The total percent of Fluoxetine peak with the degradation peak is about 93.9%.SAMPLE - SUNLIGHTFluoxetine peak:Purity Angle:0.093Match Angle:0.583Purity Threshold:0.148Match Threshold:0.825 ED. N0: 04Effective Date:APPROVED::HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.ANALYTICAL METHOD VALIDATION10 and 20mg Fluoxetine Capsules HPLC DeterminationSUNLIGHT (Cont.)5.2.Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACT was 42°C. The vials were placed in a horizontal position (4mm vials, National + Septum were used). A Dark control solution was tested. A 2%w/v quinine solution was used as the reference absorbance solution.Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of 99.5%.The difference in the absorbance in the quinine solution is 0.4227AU.Additional peak were observed at RRT of 1.5 (2.3).The total percent of Fluoxetine peak with the degradation peak is about 97.5%. STANDARD - SUNLIGHTFluoxetine peak:Purity Angle:0.067Match Angle:0.389Purity Threshold:0.134Match Threshold:0.8196.HEAT OF SOLUTION6.1.Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask. 20mL Diluent was added and the solution was sonicated for 10 minutes and made up to volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and injected into the HPLC system.Fluoxetine peak was decreased to 93.3%.SAMPLE - HEAT OF SOLUTION [105o C]Fluoxetine peak:Purity Angle:0.062Match Angle:0.460Purity Threshold:0.131Match Threshold:0.8186.2.Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL of the working solution was transferred into a suitable crucible, placed in an oven at 105°C for 2 hours, cooled to ambient temperature and injected into the HPLC system.Fluoxetine peak intensity did not decrease - 100.5%.ED. N0: 04Effective Date:APPROVED::。
超分子溶剂萃取
第42 卷第 5 期2023 年5 月Vol.42 No.5559~567分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）超分子溶剂萃取/超高效液相色谱－串联质谱法测定血浆中他克莫司含量谢以清1，2，吕悦广2，孟宪双2，雷海民1*，马强2*（1．北京中医药大学中药学院，北京102488；2．中国检验检疫科学研究院，北京100176）摘要：该文建立了血浆中免疫抑制剂他克莫司（TAC）的超分子溶剂（SUPRAS）萃取/超高效液相色谱－串联质谱分析方法。
通过单因素实验结合响应面设计对超分子溶剂组成、用量及涡旋萃取时间等关键因素进行优化后，血浆样本以正戊醇、四氢呋喃和水形成的超分子溶剂进行高效萃取。
萃取液经Waters ACQUITY UPLC BEH C18（50 mm × 2.1 mm，1.7 μm）色谱柱分离后，在电喷雾质谱正离子模式下，以多反应监测（MRM）模式对他克莫司进行测定，内标法定量。
结果表明，他克莫司在0.5 ~ 30 ng/mL质量浓度范围内的线性关系良好，相关系数（r）为0.998 6；方法检出限和定量下限分别为0.1、0.5 ng/mL；在低、中、高3个加标水平下，平均回收率（n = 3）为91.9% ~ 99.9%，相对标准偏差（RSD）为1.7% ~ 5.7%。
所建立的方法快速、灵敏、稳定，适用于血浆中他克莫司的准确测定。
关键词：他克莫司；免疫抑制剂；超分子溶剂；血浆；超高效液相色谱－串联质谱中图分类号：O657.7；R917文献标识码：A 文章编号：1004-4957（2023）05-0559-09Determination of Tacrolimus in Plasma by Supramolecular Solvent Extraction/Ultra-high Performance Liquid Chromatography－Tandem Mass SpectrometryXIE Yi-qing1，2，LÜ Yue-guang2，MENG Xian-shuang2，LEI Hai-min1*，MA Qiang2*（1．School of Chinese Materia Medica，Beijing University of Chinese Medicine，Beijing 102488，China；2．Chinese Academy of Inspection and Quarantine，Beijing 100176，China）Abstract：An analytical method for the determination of tacrolimus（TAC） in blood plasma was estab⁃lished by supramolecular solvent（SUPRAS）extraction combined with ultra-high performance liquid chromatography－tandem mass spectrometry．After optimizing the key factors such as the composition and amount of SUPRAS，and vortex extraction time through single factor experiment and response sur⁃face design，blood plasma samples were extracted efficiently with SUPRAS formed by pentanol，tetra⁃hydrofuran and water．The extract was separated on a Waters ACQUITY UPLC BEH C18column （50 mm × 2.1 mm，1.7 μm），analyzed by electrospray ionization mass spectrometry in positive ion mode under multiple reaction monitoring（MRM） mode，and quantified by internal standard method．Experimental results demonstrated that there was a good linear relationship for TAC in the concentration range of 0.5－30 ng/mL，with a correlation coefficient（r） of 0.998 6．The limit of detection（LOD）and quantitation（LOQ） were 0.1 ng/mL and 0.5 ng/mL，respectively．The average recoveries（n = 3）at low，medium and high spiked concentration levels ranged from 91.9% to 99.9%，with relative stan⁃dard deviations（RSDs） of 1.7%－5.7%．The proposed method is rapid，sensitive and stable，and it was suitable for the accurate determination of TAC in blood plasma.Key words：tacrolimus；immunosuppresive agent；supramolecular solvent；plasma；ultra-high performance liquid chromatography－tandem mass spectrometry免疫抑制剂是用于抑制机体免疫力的药物，多用于抑制肝肾移植术后的免疫反应，以及治疗变态反应性和自身免疫性疾病，如类风湿关节炎、红斑狼疮等［1－3］。
拉米夫定片
拉米夫定通用名：拉米夫定英文名：Lamivudine别名：2R-cis)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-2(1H)-pyrim idinone; 2'-Deoxy-3'-thiacytidine中文别名：拉米呋啶； (2R-顺式)-4-氨基-1-(2-羟甲基-1，3-氧硫杂环戊-5-基)-1H-嘧啶-2-酮商品名称拉米呋啶; 贺普丁产品类别: 医药原料产品含量：99%CAS 编号：134678-17-4质量标准：USP26性状：白色结晶或结晶性粉末生产厂家：葛兰素史克、安徽贝克联合制药有限公司，湖南千金湘江药业股份有限公司分子式C8H11N3O3S (2R-顺式)-4-氨基-1-(2-羟甲基-1,3-氧硫杂环戊-5-基)-1H-嘧啶-2-酮分子量229.25 CAS 登录号 134678-17-4 拉米夫定上市已经有10年，其英文名称为Lamivudine，化学名为2’3’-双脱氧-3’-硫代胞嘧啶(2’－3’deoxy-3’-thiocytidine)，又称3-TC。
在中国上市后的商品名为贺普丁。
国内外随机对照临床试验表明，每日口服100 mg 可明显抑制HBV DNA 水平，HBeAg 血清学转换率随治疗时间延长而提高，对HbeAg阳性患者治疗1、2、3、4 和5 年后HBeAg 血清转换率分别为22% 、29% 、40%、47%和50%[1-3] ；治疗前ALT 水平较高者，一般HBeAg 血清学转换率也较高[56-60] 。
长期治疗可以减轻炎症，降低肝纤维化和肝硬化的发生率[1, 2]。
随机对照临床试验表明，本药可降低肝功能失代偿和HCC（肝细胞癌）发生率[21]。
在失代偿期肝硬化患者也能改善肝功能，延长生存期[61- 63] 。
国外研究结果显示，拉米夫定治疗儿童慢性乙型肝炎的疗效与成人相似，安全性良好[64, 65] 。
化学工作者网站
化学工作者看过来，可以在线查找化学结构式！1./cgi-bin/hsrun/Distributed/HahtShop/HAHTShop.htx;start=HS_SearchCenter中，选择Search Structures，然后，就可以分别根据：Product Name 和CAS登录号，还有Molecular Formula进行查找。
在这里查到的化合物基本都有一些参数和它的结构式（Structure Image），然后将图片保存成Gif即可！当然，如果你只知道化合物的结构式，不知道叫什么，可以选择下面的Sub Structure Search ，然后 Step 1 Choose a Drawing tool. I want to Download Kekule (requires download of Kekv214.exe) Step 2 Install the plug-in you downloaded in step 1 Close this browser. Return to the directory where you saved the plug-in and double click on the file name given in step 1. Step 3 Return to this page and click the "Structure Search"button. 安装好软件即可将画好的图片粘贴上即可查找，非常方便，我用它查了一些结构式，给了我一大堆，呵呵，本人化学方面英语不是很好，一个个查看才找到，不过还好是宽带，呵呵：－）2./大家去这儿看看，查询非常简单3.【推荐】【原创】能查几万个化合物的NMR IR谱图的网址 [精华]4.能查几万个化合物的NMR IR谱图的网址 SDBS Integrated Spectral Data BaseSystem for Organic Compounds http://www.aist.go.jp/RIODB/SDBS/sdbs/owa/sdbs_sea.cre_frame_sea NIST WebBook /5.美国专利pdf全文下载的好地方 6.免费在线查合成路线/depts/chemistry/courses/toolkits/247/practice/m edialib/data/7.中科院上海有机化学研究所化学专业数据库http://202.127.145.134/tf123456/6543218.中科院上海有机化学研究所数据库（/lccdb/sjk.htm），免费注册使用，现在已经改版，性能提高许多。
分散染料产品索引号
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Disperse Yellow 211 200%分散黄5G——-——-——————---———--C.I。
Disperse Yellow 119 200％分散黄5GL—--———--—----—---—-C。
I。
Disperse Yellow 241 200%分散黄6GSL—--——--—--——-——---C.I。
Disperse Yellow 114 200％分散黄8GFF—---------—--————-C.I。
Disperse Yellow 82 200％分散红玉B—-----—--——--—----—C。
I. Disperse Red 1 200％分散红玉BD—-—--—--—--—-—---—C.I. Disperse Red 13 200%分散大红S－3GL-----—-———-—--C。
I。
Disperse Red 54 100％分散红玉S－5BL———--—--——--——C.I. Disperse Red 167 100%分散红FB——-—-—---—--——------C。
I. Disperse Red 60 200%分散红FB—-———————--——-—--—--C.I. Disperse Red 60分散红玉SE－4RB-———-—--—-———C。
TAK-875_DataSheet_MedChemExpress
Inhibitors, Agonists, Screening Libraries Data SheetBIOLOGICAL ACTIVITY:TAK–875 is a potent, selective and orally bioavailable GPR40 agonist with EC 50 of 72 nM.IC50 & Target: EC50: 72 nM (GPR40)In Vitro: TAK–875 (0.01–10 μM) produces a concentration–dependent increase in intracellular IP production in CHO–hGPR40, with EC 50of 0.072 μM. TAK–875 (0.1–10 μM) dose–dependently augments intracellular IP production in CHO cells [1]. TAK–875 (3–30 μM)concentration–dependently augments [Ca 2+]i . In the presence of 10 mM glucose, TAK–875 (0.001–10 μM) dose–dependently stimulats insulin secretion from INS–1 833/15 cells [2].In Vivo: TAK–875 (10 mg/kg, p.o.) increases plasma insulin levels in ZDF rats. TAK–875 (30 mg/kg, p.o.) improves fasting hyperglycemia without affecting fasting normoglycemia. TAK–875 at 30 mg/kg, which is a 3– to 10–fold higher dose compared with the dose that improved glucose tolerance in diabetic rats, does not alter fasting glucose levels in SD rats with normal glucose homeostasis. Likewise,TAK–875 does not significantly alter insulin secretion in SD rats with normal fasting glucose levels [1].PROTOCOL (Extracted from published papers and Only for reference)Kinase Assay:[1]INS–1 832/13 cells are suspended in RPMI medium containing 11 mM glucose and the supplementsdescribed above. These cells are seeded at a density of 2×104 cells/well in a 96–well black plate coated with poly–D–lysine, and 1%BSA and 0.1% DMSO alone (control), palmitic acid (62.5, 125, 250, 500, and 1000 μM), oleic acid (62.5, 125, 250, 500, and 1000 μM), or TAK–875 (6.25, 12.5, 25, 50, and 100 μM) is added to the plate with 1% BSA and 0.1% DMSO, followed by culture for 72 h. After the culture, caspase 3/7 activity is measured with the Apo–one homogeneous caspase 3/7 assay according to the manufacturer's instructions. Fluorescence intensity is measured at an excitation of 485 nm and an emission at 535 nm.Cell Assay: TAK–875 is dissolved in 1% BSA and 0.1% DMSO. [1]INS–1 832/13 cells are suspended in RPMI medium and seeded in a 96–well plate at a density of 2×104 cells/well; 1% BSA and 0.1% DMSO alone (control), palmitic acid (10, 100, and 1000 μM), oleic acid (10, 100, and 1000 μM), or TAK–875 (1, 10, and 100 μM) is added to the plate. After 72–h culture, medium is discarded, and cells are preincubated for 2 h with KRBH containing 1 mM glucose and 0.2% BSA at 37°C. After discarding of the preincubation buffer, KRBH containing 1 or 20 mM glucose and 0.2% BSA is added, and the plate is further incubated for 2 h. The insulin concentration in the supernatant is measured as described above. To measure intracellular insulin content, INS–1 832/13 cells are exposed to 1% BSA and 0.1% DMSO alone (control), palmitic acid (1000 μM), oleic acid (1000 μM), or TAK–875 (100 μM) with 1% BSA and 0.1% DMSO. After incubation, cells are washed once with phosphate–buffered saline, and acid–ethanol solution is added to each well, followed by sonication on ice. Intracellular insulin is extracted by overnight incubation at -30°C, followed by separation of supernatant by centrifugation at 12,000 rpm×5 min at 4°C.Animal Administration: TAK–875 is formulated in 0.5% methylcellulose.[1]At 18 weeks of age, the N–STZ–1.5 rats are fasted overnight and orally given vehicle (0.5% methylcellulose) or TAK–875 (1, 3, and 10 mg/kg). Sixty minutes later, all animals receive an oral glucoseProduct Name:TAK–875Cat. No.:HY-10480CAS No.:1000413-72-8Molecular Formula:C 29H 32O 7S Molecular Weight:524.63Target:GPR40Pathway:GPCR/G Protein Solubility:DMSO: ≥ 128 mg/mLload (1 g/kg). Blood samples are collected from the tail vein before drug administration, before glucose load (time 0), and 10, 30, 60, and 120 min after the glucose load. Plasma glucose and insulin levels are measured with an AutoAnalyzer 7080 and radioimmunoassay, respectively. To see the effects of TAK–875 on fasting normoglycemia and hyperglycemia, SD rats (8 weeks old) or ZDF and ZL rats (12 weeks old) are fasted overnight and orally given vehicle (0.5% methylcellulose), TAK–875 (10 or 30 mg/kg), nateglinide (50 mg/kg), or glibenclamide (10 mg/kg). Blood samples are collected from the tail vein before drug administration (time 0) and 0.5, 1, 2, and 3 h (SD rats) and 0.5, 1, 2, 4, and 6 h (ZDF and ZL rats) after drug administration, and plasma glucose and insulin levels are measured as described above.References:[1]. Tsujihata Y,et al. TAK–875, an orally available G protein–coupled receptor 40/free fatty acid receptor 1 agonist, enhances glucose–dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats.J Pharmacol Exp[2]. Yoshiyuki Tsujihata, et al. TAK–875, an Orally Available GPR40/FFA1 Agonist Enhances Glucose–Dependent Insulin Secretion and Improves Both Postprandial and Fasting Hyperglycemia in Type 2 Diabetic Rats. JPET July 13, 2011Caution: 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。
IFCC Aspartate Aminotransferase 检测手册说明书
ASTAspartate Aminotransferase IFCCMANUAL RX MONZAINTENDED USEFor the quantitative in vitro determination of AspartateAminotransferase (AST) in serum and plasma. This product is suitable for manual use and on the Rx Monza analyser.Cat. No. AS 1202 R1a. Buffer/Substrate 1 x 70 ml 20 x 2 ml R1b. Enzyme/Coenzyme/ 20 x 2 ml α-oxoglutarate GTIN: 05055273200416AS 1204 R1a. Buffer/Substrate 1 x 105 ml 10 x 10 ml R1b. Enzyme/Coenzyme/ 10 x 10 ml α-oxoglutarate GTIN: 05055273200423AS 1267 R1a. Buffer/Substrate 1 x 105 ml 5 x 20 ml R1b. Enzyme/Coenzyme/ 5 x 20 ml α-oxoglutarate GTIN: 05055273200430AS 2359 R1a. Buffer/Substrate 5 x 100 ml 5 x 100 ml R1b. Enzyme/Coenzyme/ 5 x 100 ml α-oxoglutarate GTIN: 05055273200454UV METHODThis is an optimised standard method according to the concentrations recommended by the IFCC.CLINICAL SIGNIFICANCE (1,2,3,4)The aminotransferases are a group of enzymes that catalyse the inter conversions of amino acids and α-oxoacids by transfer of amino groups. AST (aspartate aminotransferase or glutamate oxaloacetatetransaminase) has been found in the cytoplasm and the mitochondria of cells that have been studied. In cases of mild tissue damage, e.g. liver, the predominant form of serum AST is that from the cytoplasm, with a smaller amount coming from the mitochondria. Severe tissue damage will result in more mitochondrial enzyme being released. Elevated levels of AST can signal myocardial infarction, hepatic disease, muscular dystrophy and organ damage.Although heart muscle is found to have the most activity of the enzyme, significant activity has also been seen in the brain, liver, gastric mucosa, adipose tissue and kidneys of humans.The IFCC has now recommended (1980) standardised procedures for AST determinations including:-1. optimization of substrate concentrations.2. Employment of Tris buffers (instead of phosphate, which has beenshown to inhibit recombination of the apoenzyme with pyridoxal phosphate).3. Pre-incubation of combined buffer and serum to allow sidereactions with NADH to occur. 4. Substrate start (α-oxoglutarate)5. Optional pyridoxal phosphate activation.This is an optimised standard method according to the recommendations of the IFCC.PRINCIPLEα-oxoglutarate reacts with L-aspartate in the presence of AST to form L-glutamate plus oxaloacetate. The indicator reaction utilises the oxaloacetate for a kinetic determination of NADH consumption. AST -oxoglutarate + L-aspartate L-glutamate + oxaloacetate MDH oxaloacetate + NADH + H + L-malate + NAD +SPECIMEN COLLECTION AND PREPARATION (5) Serum:- Use serum free from haemolysis.Plasma:- EDTA or heparin can be used as the anticoagulant.Plasma should be separated from cells within one hour after collection.Specimens should be refrigerated if not used immediately:-Specimens stored longer than 3 days should be frozen at -20︒C.REAGENT COMPOSITIONContents Concentrations in the TestR1a. Buffer/Substrate Tris buffer 80 mmol/l, pH 7.5 L-aspartate 240 mmol/l R1b. Enzyme/Coenzyme/α-oxoglutarate α-oxoglutarate 12 mmol/l MDH ≥420 U/l LD ≥600 U/l NADH 0.18 mmol/lSAFETY PRECAUTIONS AND WARNINGS For in vitro diagnostic use only. Do not pipette by mouth.Exercise the normal precautions required for handling laboratory reagents.Solution R1a contains Sodium Azide. Avoid ingestion or contact with skin or mucous membranes. In case of skin contact, flush affected area with copious amounts of water. In case of contact with eyes or if ingested, seek immediate medical attention.Sodium Azide reacts with lead and copper plumbing, to form potentially explosive azides. When disposing of such reagents flush with large volumes of water to prevent azide build up. Exposed metal surfaces should be cleaned with 10% sodium hydroxide.Health and Safety data sheets available on request.The reagents must be used only for the purpose intended by suitably qualified laboratory personnel, under appropriate laboratory conditions.STABILITY AND PREPARATION OF REAGENTS R1a. Buffer/SubstrateContents ready for use. Stable up to the expiry date when stored at +2 to +8︒C.R1b. Enzyme/Coenzyme/α-oxoglutarate Reconstitute one vial of Enzyme/Coenzyme/α-oxoglutarate R1b with the appropriate volume of Buffer/Substrate R1a: 2 ml for the 20 x 2 ml kit (AS 1202) 10 ml for the 10 x 10 ml kit (AS 1204) 20 ml for the 5 x 20 ml kit (AS 1267) Stable for 14 days at +2 to +8︒C or 24 hours at +15 to +25︒C. Cat. AS 2359 5 x 100 mlReconstitute one vial of Enzyme/Coenzyme/α-oxoglutarate R1b with a portion of Buffer/Substrate R1a and then transfer the entire contents to bottle R1a rinsing bottle R1b several times. Stable for 14 days at +2 to +8︒C or 24 hours at +15 to +25︒C.MATERIALS PROVIDED Buffer/SubstrateEnzyme/Coenzyme/ -oxoglutarateMATERIALS REQUIRED BUT NOT PROVIDEDRandox Assayed Multisera Level 2 (Cat. No. HN 1530) and Level 3 (Cat. No. HE 1532)Randox Calibration Serum Level 3 (Cat. No. CAL 2351) RX series Saline (Cat. No. SA 3854)PROCEDUREAspirate fresh ddH 2O and perform a new Gain Calibration in flow cell mode. Select AST in the Run Test screen and carry out a water blank as instructed.Pipette into a test tube:Sample 0.05 ml Reagent 0.5 mlMix and aspirate into the Rx Monza.CALIBRATION FOR RX MONZAThe use of Saline and Randox Calibration Serum Level 3 isrecommended for calibration. Calibration is recommended with change of reagent lot or as indicated by quality control procedures.FOR MANUAL USEWavelength: 340 nm (Hg 334 nm or Hg 365 nm) Cuvette: 1 cm light path Temperature: 25/30/37︒C Measurement: against airPipette into cuvette: Macro MicroSample 0.2 ml 0.1 ml Enzyme/Coenzyme/ α-oxoglutarate R1 2.0 ml 1.0 mlMix, read initial absorbance after 1 minute. Read again after 1, 2 and 3 minutes. Note: If the absorbance change per minute is between 0.11 and 0.16 at 340/Hg 334 nm 0.06 and 0.08 at Hg 365 nmuse only the values for the first 2 minutes for the calculation.MANUAL CALCULATIONTo calculate the AST activity, use the following formulae:U/l = 1746 x A 340 nm/min U/l = 1780 x A Hg 334 nm/min U/l = 3235 x A Hg 365 nm/minSTANDARDISATIONRandox Calibration Serum Level 3 is traceable to AST reference material JSCC TS01.QUALITY CONTROLRandox Assayed Multisera, Level 2 and Level 3 are recommended for daily quality control. Two levels of controls should be assayed at least once a day. Values obtained should fall within a specified range. If these values fall outside the range and repetition excludes error the following steps should be taken:1. Check instrument settings and light source.2. Check cleanliness of all equipment in use.3. Check water. Contaminants, i.e. bacterial growth, maycontribute to inaccurate results. 4. Check reaction temperature.5. Check expiry date of kit and contents.6. Contact Randox Laboratories Customer Technical Services, Northern Ireland +44 (0) 28 9445 1070.SPECIFICITY/INTERFERENCE (6,7)Gross haemolysis will produce falsely elevated test results. The effects of various drugs on AST activity should be taken intoconsideration in the case of patients receiving large doses of drugs.The analytes below were tested up to the following levels and were found not to interfere: Haemoglobin 250 mg/dl Free Bilirubin 25 mg/dl Conjugate Bilirubin 25 mg/dl Triglycerides 1000 mg/dlIntralipid ® 200 mg/dlA list of substances and conditions known to effect AST activity in vivo is given by both Young et al and Friedman et al. Norepresentation is made by Randox Laboratories Ltd regarding the completeness of these lists and the accuracy of the information contained therein.NORMAL VALUES IN SERUM (8,9) +25︒C +30︒C +37︒C Men up to 18 U/l up to 25 U/l up to 37 U/l Women up to 15 U/l up to 21 U/l up to 31 U/lIt is recommended that each laboratory establish its own reference range to reflect the age, sex, diet and geographical location of the population.SPECIFIC PERFORMANCE CHARACTERISTICS The following performance data were obtained using an Rx Monza analyser running at +37o C.LINEARITYThis method is linear up to 562 U/l. If the sample concentration exceeds this value, dilute the sample 1+9 with 0.9% NaCl solution and re-assay. Multiply the result by 10.SENSITIVITYThe minimum detectable concentration of AST with an acceptable level of precision was determined as 9.3 U/l.PRECISIONIntra AssayLevel 2 Level 3Mean (U/l) 35.6 153SD 1.66 1.47CV(%) 4.65 0.96n 20 20Inter AssayLevel 2 Level 3Mean (U/l) 35.6 153SD 1.77 7.10CV(%) 4.96 4.63n 20 20CORRELATIONThis method (Y) was compared with another commerciallyavailable method (X) and the following linear regression equationobtained:Y = 1.07X + 4.9and a correlation coefficient of r = 0.997543 patient samples were analysed spanning the range 28 to 559U/l.REFERENCES1. Wroblewski F, La Due J.S: Ann Intern Med. 1956; 45: 801.2. Wroblewski F, La Due J.S: Proc Soc Exp Biol Med 1956;91: 569.3. Bergmeyer HU, Bowers GN Jr, et al: Clin Chem 1977; 23:887.4. Bergmeyer HU, Bowers GN Jr, et al: J.Clin Chem ClinBiochem 1980; 18: 521-534.5. Tietz N W: Fundamentals of Clinical Chemistry ed 3.Philadelphia, WB Saunders Co. 1987, pg 372.6. Young D S, et al: Clin Chem 1975, 21; No5.7. Friedman RB, et al: Clin Chem 1980, 26; No4.8. Wallnofer H, Schmidt.E, Schmidt FW, eds: Synopsis derLeberkrankheiten Stuttgart, Georg Thieme Verlag, 1974.9. Thefeld W, et al: Dtsch Med Wschr 1974; 99: 343.Revised 26 Apr 16 biRev. 003THIS PAGE IS INTENTIONALLY BLANK。
三氟乙酸脱苄基O-debenzylation_of_ortho-substituted_phenols_with_trifluoroacetic_acid
Mild,efficient and rapid O-debenzylation of ortho -substituted phenols with trifluoroacetic acidSteven Fletcher *,Patrick T.Gunning *Department of Chemistry,University of Toronto,Mississauga,ON L5L 1C6,Canadaa r t i c l e i n f o Article history:Received 21May 2008Revised 2June 2008Accepted 4June 2008Available online 10June 2008a b s t r a c tThe mild and efficient deblocking of aryl benzyl ethers with TFA is reported.Cleavage was fastest with ortho -electron-withdrawing groups on the phenolic ring,which we have attributed to a proton chelation effect,furnishing the deprotected phenols in excellent yields.The corresponding para -methoxybenzyl,allyl and iso -propyl ethers were also cleanly removed under these conditions.In addition,the selective aryl benzyl ether debenzylation in the presence of benzyl ester,Cbz carbamate and Boc carbamate func-tionalities was also observed.Crown Copyright Ó2008Published by Elsevier Ltd.All rights reserved.Phosphotyrosines feature in the design of inhibitors of several protein targets,including protein tyrosine phosphatase 1B (PTP1B).1However,these moieties suffer from hydrolytic lability to cellular phosphatases and poor cell penetration due to the asso-ciated dianionic charge.1To address these issues,salicylic acid derivatives (and closely-related analogues)have become popular mimetics of phosphotyrosine in small molecule inhibitors.1–5Turk-son et al.have recently reported on NSC74859(1),a potent,sali-cylic acid-based inhibitor of the oncogenic protein Stat3.6As part of our structure–activity relationship (SAR)studies on NSC74859(1),we sought to debenzylate both the phenol ether and benzoate ester in 2without reducing the aryl-bromide bond,a common undesired side reaction that occurs with hydrogen gas and Pd/C catalyst.7O -Benzyl-protected phenols are known to undergo debenzyla-tion with trifluoroacetic acid (TFA)8by an initial protonation of the weakly basic phenol oxygen,although additives such as strongorganic acids (e.g.,trifluoromethanesulfonic acid 9)or a large excess of nucleophilic scavenger (e.g.,thioanisole,which accelerates the reaction by a ‘push–pull’mechanism 10)are typically required.Re-cent work by Ploypradith et al.describes the mild deprotection of aromatic ethers with sub-stoichiometric para -toluenesulfonic acid on solid support.11In a special case,O -benzyl-protected ortho -nitrophenol was cleaved rapidly (<5min)with neat TFA,12which we considered was due to the ability of the substrate to chelate a proton since the structurally-similar ortho -hydroxybenzoates (salicylates)are well-known to chelate copper ions and iron ions.We reasoned that 2(and indeed 3)may similarly undergo acceler-ated debenzylation with TFA.In fact,as shown in Scheme 1,treat-ment of 2(or 3)with a 1:1mixture of TFA/toluene led to rapid debenzylation (5min for 2;1h for 3)in 91%yield for 2(or 85%yield for 3).In this Letter,we will explore the structural require-ments of the phenol component that increase the lability of the O -benzyl phenol ether bond in the presence of TFA.In addition,0040-4039/$-see front matter Crown Copyright Ó2008Published by Elsevier Ltd.All rights reserved.*Tel.:+19058285354;fax:+19058285425(P.T.G.).E-mail addresses:steven.fletcher@utoronto.ca (S.Fletcher),patrick.gunning@utoronto.ca (P.T.Gunning).Tetrahedron Letters 49(2008)4817–4819Contents lists available at ScienceDirectTetrahedron Lettersj o ur na l h om e pa ge :w w w.e ls e v ie r.c o m/lo c at e/t et l e twe will explore the selectivity of this mild debenzylation tech-nique with respect to other aromatic ethers and examine the sta-bility of other benzyl-based protecting groups to these reaction conditions.A series of 12O -benzyl-protected phenols was prepared by standard procedures in near quantitative yields.Each of these ethers was then deprotected with a 1:1mixture of TFA/toluene;our observations are summarized in Table 1.In certain cases,O ?C benzyl migration (Friedel–Crafts reaction)by-products (610%)were occasionally inseparable from the product by silica gel flash column chromatography.Thus,several benzyl cation cap-tors were investigated for their abilities to improve yields and puri-ties of the debenzylation reactions.Three to ten equivalents of p -cresol,anisole and triethylsilane were employed,but these exerted little effects on reducing by-product formation.Conversely,we dis-covered that including the more nucleophilic scavenger thioanisole as an additive to the co-solvent toluene typically,after silica gel flash column chromatography,furnished products in P 95%puri-ties (and higher yields),as judged by 1H NMR.Nevertheless,we envisaged any Friedel–Crafts impurities would be more readily separable on slightly more complex aryl benzyl ethers,as we ob-served with the substrates shown in Scheme 1and Tables 3and 4(>99%purities (1H NMR)in each case).Whilst likely leading to even higher yields and purities,large excesses of thioanisole (50equiv)are also known to accelerate TFA-mediated debenzyla-tion.10However,in our hands just 3equiv of thioanisole had little effect on the rate of debenzylation,allowing us to attribute the deprotection rates solely to the structure of the phenol.Electron-rich phenols are good scavengers of benzyl cations,13and since preliminary experiments with electron-rich phenols generated complex mixtures of Friedel–Crafts by-products under these deb-enzylation conditions,we chose to investigate only electron-poor phenols in this study.O -Benzyl-protected phenols with p -ortho -electron-withdraw-ing groups (6a ,6b ,6d ,6f )were swiftly (several in less than 3h cf.24h for unsubstituted phenol 6l )and cleanly debenzylated,with less than 5%of the undesired C-benzylated phenol by-prod-ucts.In contrast,meta -and para -electron-withdrawing groups slo-wed down the debenzylation (e.g.,entries 6g and 6h ),relative to the control compound 6l ,which itself could only be obtained in moderate purity by this method.The r -withdrawing (and p -donating)bromophenols 6i –k were insufficiently deactivated to benzyl cation scavenging and were contaminated with several by-products.Importantly,n -butyl benzyl ether 8was unaffected by TFA under the reaction conditions,indicating this procedure is selective for aryl benzyl ethers.In addition,the results in Table 1suggest that this procedure is suitable only for phenols substituted with p -electron-withdrawing groups.Since the debenzylation mechanism with TFA proceeds via an initial protonation of the phenol ether oxygen,the more available the ether oxygen lone pairs are,the faster the reaction will be.Hence,the slower reaction times for the phenols bearing meta -and para -electron-withdrawing groups make sense,although this is not true for the ortho -functionalized aryl benzyl ethers.As hypothesized for the bis-benzyl salicylate derivative 2earlier,we considered these ortho -substituted phenols were capable of chelat-ing the acidic hydrogen atom from TFA which therein facilitated the acid-mediated debenzylation via a six-membered cyclic inter-mediate,as proposed in Scheme 2.A similar chelation intermediate has been put forward by Baldwin and Haraldsson to account for the Lewis acid MgBr 2-mediated debenzylation of aromatic benzyl ethers ortho to an aldehyde group.14Accordingly,to test this hypothesis we expanded this series of ortho -substituted aryl benzyl ethers,and the results from their deb-enzylation reactions with TFA are summarized in Table 2.These substrates have been listed in order of increasing approximateTable 1TFA-mediated debenzylation of O -benzyl-protected phenols aTFAtolueneOBnROHR67Substrate RTime (h)b Yield c (%)6a o -CO 2Me,m d -NHAc 5min 936b o -CO 2Me 5min 946c p -CO 2Me 36e 63(85f )6d o -CO 2Bn 5min 936e p -CO 2Bn 36e 58(79f )6f o -NO 23976g m -NO 236e 75(98f )6h p -NO 236e 66(98f )6i o -Br 16—g 6j m -Br 30—g 6k p -Br 36—g 6lH 24—gn -BuOBn (8)—24No reactionaThe reaction was carried out with 6(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt,with 3equiv of thioanisole.bTime taken for all starting material to be consumed.cIsolated yield after silica gel flash column chromatography.dmeta to phenol oxygen AND para to ester.eReaction was slow and incomplete after 3days.fYield based on recovered starting material.gComplex mixture of products.Table 2TFA-mediated debenzylation of O -benzyl-protected,ortho -substituted phenols aTFA tolueneOBnOH67RRSubstrate R p K aH b Time c (h)Yield d (%)Relative rate 6m CO 2NH 2À2248316n CHO À7 3.594e 6.96o CO 2H À8191246b CO 2Me À8.55min 942886d CO 2Bn À8.55min 932886p CN À10>4851(95f )—6f NO 2À1239786i Br —16—g 1.56lH—24—g1aThe reaction was carried out with 6(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt,with 3equiv of thioanisole.bApproximate p K aH of conjugate acid of R group.15cTime taken for all starting material to be consumed.dIsolated yield after silica gel flash column chromatography.eIncluding thioanisole in the deprotection of 6n led to further by-products,thus no scavenger was used and compound 7n could be obtained in only 90%purity.fYield based on recovered starting material.gComplex mixture of products.4818S.Fletcher,P.T.Gunning /Tetrahedron Letters 49(2008)4817–4819acidity of the conjugate acid (decreasing p K aH )of the ortho -elec-tron-withdrawing substituent.15There appears to be an optimal p K aH of around À8.5,that is exhibited by carboxylic esters,which lead to the fastest rate of debenzylation with TFA.In an approxi-mate bell-shaped distribution of reaction rate versus ortho -substi-tuent p K aH —that was interrupted only by ortho -cyanophenol 6p —protonatable groups with p K aH ’s <À8.5or >À8.5were less effective at accelerating the TFA-mediated debenzylation.These data concur with our chelation hypothesis:groups that are too ba-sic bind more strongly to the TFA proton making it less available for sharing with,and ultimately releasing to,the phenol ether oxygen;groups that are weakly basic do not bind the TFA proton as well,leading to reduced chelation and hence less rate enhancement.The anomalous result for ortho -cyanophenol 6p was anticipated since this compound was selected as a negative control.Phenol 6p is geometrically incapable of chelating a proton,because the lin-ear,sp -hybridized nitrile functionality directs its basic nitrogen atom (p K aH %À10)away from the phenol oxygen.As predicted,there was no rate enhancement for the TFA-mediated debenzyla-tion of 6p relative to phenol 6l .In fact,6p was only slowly deben-zylated,at a rate that was comparable with the m -nitro and p -nitro derivatives 6g and 6h ,respectively.We next wanted to investigate the selectivity for the deprotec-tion of the benzyl group over other phenol protecting groups.Accordingly,the benzyl group in salicylate derivative 9a was varied with para -methoxybenzyl (PMB;9b ),methyl (9c ),allyl (9d )and iso -propyl (i -Pr;9e ).These substrates were then debenzylated with a 1:1mixture of TFA/toluene;our findings are reported in Table 3.Any impurities this time were minor and readily separable from the products,eliminating the need for the additive thioanisole.The relative rates at which these protecting groups were removed was para -methoxybenzyl >benzyl >allyl >iso -propyl )methyl,which reflects the stability of the carbocations.These data suggest that in salicylates such as 9,the benzyl phenol protecting group (R =Bn)can be removed with TFA in the presence of the corres-ponding allyl,iso -propyl and methyl ethers.Finally,we explored the selectivity of this mild debenzylation technique over other benzyl-based protecting groups,as shown in Table 4.As the results demonstrate,it was possible to deblock the O -benzyl ether in the presence of a benzyl ester (6d )and in the presence of a benzyl carbamate (11b ),thereby increasing the orthogonality of O -benzyl phenol ethers of salicylate derivatives.Interestingly,it was even possible to cleave the benzyl group in 11c with TFA in the presence of an N -Boc-protected aniline.In summary,we have presented the mild,efficient and rapid deblocking of ortho -substituted aryl benzyl ethers with TFA.Deb-enzylation was fastest when the ortho group was a carboxylic ester,which we have attributed to a proton chelation effect.Other ortho groups that accelerated the TFA-mediated debenzylation included carboxylic acid,aldehyde and nitro.In addition,we have shown that in such ortho -functionalized phenols,benzyl could be removed in the presence of the corresponding iso -propyl,allyl and methyl ethers.Moreover,the benzyl ether could be selectively cleaved in the presence of benzyl ester,Cbz carbamate and Boc carbamate functionalities.AcknowledgementsThe authors gratefully acknowledge financial support for this work from the Canadian Foundation of Innovation and the Univer-sity of Toronto (Connaught Foundation).References and notes1.Zhang,S.;Zhang,Z.-Y.Drug Discov.Today 2007,12,373–381.2.(a)Pei,Z.;Li,X.;Liu,G.;Abad-Zapatero,C.;Lubben,T.;Zhang,T.;Ballaron,S.J.;Hutchins,C.W.;Trevillyana,J.M.;Jirouseka,M.R.Bioorg.Med.Chem.Lett.2003,13,3129–3132;(b)Xin,Z.;Liu,G.;Abad-Zapatero,C.;Pei,Z.;Szczepankiewicz,B.G.;Li,X.;Zhang,T.;Hutchins,C.W.;Hajduk,P.J.;Ballaron,S.J.;Stashko,M.A.;Lubben,T.H.;Trevillyana,J.M.;Jirouseka,M.R.Bioorg.Med.Chem.Lett.2003,13,3947–3950.3.Tautz,L.;Bruckner,S.;Sareth,S.;Alonso,A.;Bogetz,J.;Bottini,N.;Pellecchia,M.;Mustelin,T.J.Biol.Chem.2005,280,9400–9408.4.Shrestha,S.;Bhattarai,B.R.;Chang,K.J.;Leea,K.-H.;Choa,H.Bioorg.Med.Chem.Lett.2007,17,2760–2764.5.Liljebris,C.;Larsen,S.D.;Ogg,D.;Palazuk,B.J.;Bleasdale,J.E.J.Med.Chem.2002,45,1785–1798.6.Siddiquee,K.;Zhang,S.;Guida,W.C.;Blaskovich,M.A.;Greedy,B.;Lawrence,H.R.;Yip,M.L.R.;Jove,R.;Laughlin,M.M.;Lawrence,N.J.;Sebti,S.M.;Turkson,J.Proc.Natl.Acad.Sci.U.S.A.2007,104,7391–7396.7.Pandey,P.N.;Purkayastha,M.L.Synthesis 1982,876–878.8.(a)Greene,T.W.;Wuts,P.G.M.Protective Groups in Organic Synthesis ,3rd ed.;John Wiley &Sons:New York,1999;(b)Kocienski,P.J.Protecting Groups ,3rd ed.;Georg Thieme:Stuttgart,Germany,2003.9.Kiso,Y.;Isawa,H.;Kitagawa,K.;Akita,T.Chem.Pharm.Bull.1978,26,2562–2564.10.Kiso,Y.;Ukawa,K.;Nakamura,S.;Ito,K.;Akita,T.Chem.Pharm.Bull.1980,28,673–676.11.Ploypradith,P.;Cheryklin,P.;Niyomtham,N.;Bertoni,D.R.;Ruchirawat,.Lett.2007,9,2637–2640.12.Marsh,J.P.,Jr.;Goodman,.Chem.1965,30,2491–2492.13.(a)Eberle,A.N.J.Chem.Soc.,Perkin Trans.11986,361–367;(b)Bodanszky,M.;Tolle,J.C.;Deshmane,S.S.;Bodanszky,A.Int.J.Pept.Protein Res.1978,12,57–68.14.Haraldsson,G.G.;Baldwin,J.E.Tetrahedron 1997,53,215–224.15.(a)Ionization Constants of Organic Acids in Solution ;Serjeant,E.P.,Dempsey,B.,Eds.IUPAC Chemical Data Series No.23;Pergamon Press:Oxford,UK,1979;(b)see also:/labs/evans/pdf/evans_pKa_table.pdf .Table 3TFA-mediated deprotection of O-blocked phenol ether derivatives of methyl 4-acetamidosalicylate aTFAtolueneNHAcNHAcORO OMeOH OMeO 910Substrate R Time b (h)Yield c (%)9a Bn 5min 919b PMB 2min 909c Me 480d 9d Allyl 20919ei -Pr3692aThe reaction was carried out with 9(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt.bTime taken for all starting material to be consumed.cIsolated yield after silica gel flash column chromatography.dOnly starting material remained after 48h,at which point the reaction was aborted.Table 4Selectivity investigation into the TFA-mediated debenzylation of aryl benzyl ethers aTFA tolueneOBnOH2Bn2Bn1112RRSubstrate R Yield b (%)6d c H 9311a NHAc 9211b NHCbz 9311c dNHBoc54aThe reaction was carried out with 11(0.5mmol)in a 1:1mixture of TFA/toluene (5ml)at rt for 5min,then all solvents were evaporated.bIsolated yield after silica gel flash column chromatography.cFor compound 6d ,3equiv of thioanisole were also used.dAfter 5min,the reaction mixture was diluted with CH 2Cl 2and then immedi-ately neutralized with 1M NaOH.The organic layer was then separated and evaporated.S.Fletcher,P.T.Gunning /Tetrahedron Letters 49(2008)4817–48194819。
(2020)食品添加剂使用卫生标准
食品添加剂使用卫生标准食品添加剂使用卫生标准【GB 2760—1996】前言本标准代替GB 2760—86《食品添加剂使用卫生标准》及GB 2760—86《食品添加剂使用卫生标准(1988年、1989年、1990年的增补品种)》。
在修订GB 2760—86时，食品添加剂的类别是采用了GB 12493—90《食品添加剂分类和代码》及GB/T 14156—93《食品用香料分类与编码》的分类及代码、编码，并增加美国香味料和萃取物制造者协会(FEMA)编号，按英文字母次序排列。
原标准中许多食品名称不统一，现根据有关国家标准及术语进行了修改。
本标准于1977年首次发布，1996年12月进行第三次修订。
本标准自实施之日起，同时代替2760—86。
本标准的附录A、附录B、附录C都是标准的附录；本标准的附录D是提示的附录。
本标准由全国食品添加剂标准化技术委员会提出。
本标准由卫生部食品卫生监督检验所负责起草。
本标准主要起草人高鹤娟、陈瑶君、郑鹏然、戴滢、袁亦丞、周树南、戴寅。
本标准由卫生部委托技术归口单位卫生部食品卫生监督检验所负责解释。
1 范围本标准规定了食品添加剂的品种、使用范围及最大使用量。
本标准适用于所有使用食品添加剂的生产经营者。
2 引用标准下列标准所包含的条文，通过在本标准中引用而构成为本标准的条文。
本标准出版时，所示版本均为有效。
所有标准都会被修订，使用本标准的各方应探讨使用下列标准最新版本的可能性。
GB 12493—90 食品添加剂分类和代码GB/T 14156—93 食品用香料分类与编码GB 14880—94 食品营养强化剂使用卫生标准3 食品添加剂品种、使用范围、最大使用量食品添加剂品种、使用范围、最大使用量应符合表1规定。
*下表已经根据GB2760-1996《食品添加剂使用卫生标准》第1号修改单进行了修改。
该修改单经国家质量技术监督局于1999年11月15日以质技监标函[1999]189号文批准，自2000年2月1日起实施。
MEKC血样中药物,外文翻译
MEKC血样中药物.第1页原始文件沃尔夫冈·BuchbergerÆ马蒂亚斯Ferdig鲁道夫·索默Æ翠THANH VO 雷帕霉素在人体血液中的微量分析胶束电动色谱收稿日期：2004年3月16日/日期：11 2004/5月接受日期：2004年5月18号/发表时间：2004年7月20日Ó施普林格出版社2004年摘要毛细管电泳法与UV检测波长为278 nm的分析已经开发在人类的免疫抑制剂雷帕霉素（西罗莫司）血微克每升的水平低。
分离有在酸性载体电解质含有已取得十二烷基硫酸钠和30％（体积/体积）腈。
为样品净化和富集，离线固固相萃取的步骤中使用的基于二氧化硅的反相材料和毛细管聚焦技术采用。
后者允许注射增加扩大样本量，而不会过度带。
虽然这种新方法是不敏感，比现有的质谱联用液相色谱程序法，它是完全适合常规分析的RA-pamycin血液中这种药物治疗的患者。
最后，但并非最不重要的，低的成本使一个有吸引力的建立的方法替代。
关键词雷帕霉素Æ胶束电动血色谱Æ分析介绍雷帕霉素（西罗莫司）是三烯大环内酯类抗生素具有抗真菌，消炎，抗肿瘤和这是目前使用的免疫抑制特性用于预防器官移植排斥反应的反式种植园。
雷帕霉素的结构图给出。
1。
雷怕霉素已被证明是阻止T 细胞活化和扩散以及激活P70 S6文FK-506结合激酶，表现出很强的结合蛋白质。
它也抑制活性的蛋白质哺乳动物雷帕霉素靶蛋白（mTOR）的，该功能蒸发散在促进肿瘤生长的信号转导通路。
雷帕霉素结合到受体蛋白质（FKBP12），和的rapamycin/FKBP12复杂的结合mTOR的并阻止mTOR的互动与目标蛋白质在这一信号通路。
对于栽种的器官的患者是必不可少的监测雷帕霉素在他们的血液中的水平。
决定雷帕霉素在血液中的中断可以确定迄今已完成高效液相色谱紫外检测器[1-4]，最近由高效液相色谱法，连字符和质谱（MS）[5-8]。
AZD7545_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 :AZD7545Catalog No. :HY-16082CAS No. :252017-04-21.2 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 mixtureGHS Classification in accordance with 29 CFR 1910 (OSHA HCS)Acute toxicity, Oral (Category 4),H302Acute aquatic toxicity (Category 1),H400Chronic aquatic toxicity (Category 1),H4102.2 GHS Label elements, including precautionary statementsPictogramSignal word WarningHazard statement(s)H302 Harmful if swallowed.H410 Very toxic to aquatic life with long lasting effects.Precautionary statement(s)P264 Wash skin thoroughly after handling.P270 Do not eat, drink or smoke when using this product.P273 Avoid release to the environment.P301 + P312 IF SWALLOWED: Call a POISON CENTER or doctor/ physician if you feel unwell.P330 Rinse mouth.P391 Collect spillage.P501 Dispose of contents/ container to an approved waste disposal plant.2.3 Other hazardsNone.3. COMPOSITION/INFORMATION ON INGREDIENTS3.1 SubstancesSynonyms:AZD 7545; AZD⁻7545Formula:C19H18ClF3N2O5SMolecular Weight:478.87CAS No. :252017-04-24. 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 White to off-white (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.IMDGUN number: 3077Class: 9Packing group: IIIEMS-No: F-A, S-FProper shipping name: ENVIRONMENTALLY HAZARDOUS SUBSTANCE, SOLID, N.O.S.Marine pollutant: Marine pollutantIATAUN number: 3077Class: 9Packing group: IIIProper shipping name: Environmentally hazardous substance, solid, n.o.s.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。
OECD Test No. 460 Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irrit
OECD/OCDE460Adopted: 2 October 2012© OECD, (2012)You are free to use this material for personal, non-commercial purposes without seeking prior consent from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written permission from the OECD.OECD GUIDELINE FOR THE TESTING OF CHEMICALS Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe IrritantsINTRODUCTION1. The Fluorescein Leakage (FL) test method is an in vitro test method that can be used under certain circumstances and with specific limitations to classify chemicals (substances and mixtures) as ocular corrosives and severe irritants, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Category 1), the European Union (EU) Regulation on Classification, Labelling and Packaging of Substances and Mixtures (CLP) (Category 1), and the U.S. Environmental Protection Agency (EPA) (Category I) (1) (2) (3). For the purpose of this Test Guideline, severe irritants are defined as chemicals that cause tissue damage in the eye following test substance administration that is not reversible within 21 days or causes serious physical decay of vision, while ocular corrosives are chemicals that cause irreversible tissue damage to the eye. These chemicals are classified as UN GHS Category 1, EU CLP Category 1, or U.S. EPA Category I.2. While the FL test method is not considered valid as a complete replacement for the in vivo rabbit eye test, the FL is recommended for use as part of a tiered testing strategy for regulatory classification and labelling. Thus, the FL is recommended as an initial step within a Top-Down approach to identify ocular corrosives/severe irritants, specifically for limited types of chemicals (i.e. water soluble substances and mixtures) (4)(5).3. It is currently generally accepted that, in the foreseeable future, no single in vitro eye irritation test will be able to replace the in vivo eye test (TG 405 (6)) to predict across the full range of irritation for different chemical classes. However, strategic combinations of several alternative test methods within a (tiered) testing strategy may be able to replace the in vivo eye test (5). The Top-Down approach (5) is designed to be used when, based on existing information, a chemical is expected to have high irritancy potential.Based on the prediction model detailed in paragraph 35, the FL test method can identify Category 1; EU CLP Category 1; U.S. EPA Category I) without any further testing. The same is assumed for mixtures although mixtures were not used in the validation. Therefore, the FL test method may be used to determine the eye irritancy/corrosivity of chemicals, following the460OECD/OCDEsequential testing strategy of TG 405 (6). However, a chemical that is not predicted as ocular corrosive or severe irritant with the FL test method would need to be tested in one or more additional test methods (in vitro and/or in vivo) that are capable of accurately identifying i) chemicals that are in vitro false negative ocular corrosives/severe irritants in the FL (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I); ii) chemicals that are not classified for eye corrosion/irritation (UN GHS No Category; EU CLP No Category; U.S. EPA Category IV); and/or iii) chemicals that are moderate/mild eye irritants (UN GHS Categories 2A and 2B; EU CLP Category 2; U.S. EPA Categories II and III).5. The purpose of this Test Guideline is to describe the procedures used to evaluate the potential ocular corrosivity or severe irritancy of a test substance as measured by its ability to induce damage to an impermeable confluent epithelial monolayer. The integrity of trans-epithelial permeability is a major function of an epithelium such as that found in the conjunctiva and the cornea. Trans-epithelial permeability is controlled by various tight junctions. Increasing the permeability of the corneal epithelium in vivo has been shown to correlate with the level of inflammation and surface damage observed as eye irritation develops.6. In the FL test method, toxic effects after a short exposure time to the test substance are measured by an increase in permeability of sodium fluorescein through the epithelial monolayer of Madin-Darby Canine Kidney (MDCK) cells cultured on permeable inserts. The amount of fluorescein leakage that occurs is proportional to the chemical-induced damage to the tight junctions, desmosomal junctions and cell membranes, and can be used to estimate the ocular toxicity potential of a test substance. Annex I provides a diagram of MDCK cells grown on an insert membrane for the FL test method.7. Definitions are provided in Annex II.INITIAL CONSIDERATIONS AND LIMITATIONS8. This Test Guideline is based on the INVITTOX protocol No. 71 (7) that has been evaluated in an international validation study by the European Centre for the Validation of Alternative Methods (ECVAM) (8), in collaboration with the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the Japanese Center for the Validation of Alternative Methods (JaCVAM).9. The FL test method is not recommended for the identification of chemicals which should be classified as mild/moderate irritants or of chemicals which should not be classified for ocular irritation (substances and mixtures) (i.e. GHS Cat. 2A/2B, no category; EU CLP Cat. 2, no category; US EPA Cat. II/III/IV), as demonstrated by the validation study (4) (8).10. The test method is only applicable to water soluble chemicals (substances and mixtures). The ocular severe irritation potential of chemicals that are water soluble and/or where the toxic effect is not affected by dilution is generally predicted accurately using the FL test method (8). To categorise a chemical as water soluble, under experimental conditions, it should be soluble in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, Hanks’ Buffered Salt Solution (HBSS) at a concentration ≥ 250 mg/mL (one dose above the cut-off of 100 mg/mL). However, if the test substance is soluble below the concentration 100 mg/mL,2© OECD, (2012)OECD/OCDE 460 but already induces a FL induction of 20 % at that concentration (meaning FL20 < 100 mg/mL), it can still be classified as GHS Cat. 1 or EPA Cat. 1.11. The identified limitations for this test method exclude strong acids and bases, cell fixatives and highly volatile chemicals from the applicability domain. These chemicals have mechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponification or specific reactive chemistries. Other identified limitations for this method are based upon the results for the predictive capacity for coloured and viscous test substance (8). It is suggested that both types of chemicals are difficult to remove from the monolayer following the short exposure period and that predictivity of the test method could be improved if a higher number of washing steps was used. Solid chemicals suspended in liquid have the propensity to precipitate out and the final concentration to cells can be difficult to determine. When substances within these chemical and physical classes are excluded from the database, the accuracy of FL across the EU, EPA, and GHS classification systems is substantially improved (8).12. Based on the purpose of this test method (i.e. to identify ocular corrosives/severe irritants only), false negative rates (see Paragraph 13) are not critical since such substances would be subsequently tested with other adequately validated in vitro tests or in rabbits, depending on regulatory requirements, using a sequential testing strategy in a weight of evidence approach (6) (see also paragraphs 3 and 4).13. Other identified limitations of the FL test method are based on false negative and false positive rates. When used as an initial step within a Top-Down approach to identify water soluble ocular corrosive/severe irritant substances and mixtures (UN GHS Category 1; EU CLP Category 1; U.S. EPA Category I), the false positive rate for the FL test method ranged from 7% (7/103; UN GHS and EU CLP) to 9% (9/99; U.S. EPA) and the false negative rate ranged from 54% (15/28; U.S. EPA) to 56% (27/48; UN GHS and EU CLP) when compared to in vivo results. Chemical groups showing false positive and/or false negative results in the FL test method are not defined here.14. Certain technical limitations are specific to the MDCK cell culture. The tight junctions that block the passage of the sodium-fluorescein dye through the monolayer are increasingly compromised with increasing cell passage number. Incomplete formation of the tight junctions results in increased FL in the non-treated control. Therefore, a defined permissible maximal leakage in the non-treated controls is important (see paragraph 38: 0% leakage). As with all in vitro assays there is the potential for the cells to become transformed over time, thus it is vital that passage number ranges for the assays are stated.15. The current applicability domain might be increased in some cases, but only after analyzing an expanded data set of studied test substances, preferably acquired through testing (4). This Test Guideline will be updated accordingly as new information and data are considered.16. For any laboratory initially establishing this assay, the proficiency chemicals provided in Annex III should be used. Laboratories can use these chemicals to demonstrate their technical competence in performing the FL test method prior to submitting FL assay data for regulatory hazard classification purposes.PRINCIPLE OF THE TEST3© OECD, (2012)460OECD/OCDE17. The FL test method is a cytotoxicity and cell-function based in vitro assay that is performed on a confluent monolayer of MDCK CB997 tubular epithelial cells that are grown on semi-permeable inserts and model the non-proliferating state of the in vivo corneal epithelium. The MDCK cell line is well established and forms tight junctions and desmosomal junctions similar to those found on the apical side of conjunctival and corneal epithelia. Tight and desmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal epithelium. Loss of trans-epithelial impermeability, due to damaged tight junctions and desmosomal junctions, is one of the early events in chemical-induced ocular irritation.18. The test substance is applied to the confluent layer of cells grown on the apical side of the insert. A short 1 min exposure is routinely used to reflect the normal clearance rate in human exposures. An advantage of the short exposure period is that water-based substances and mixtures can be tested neat, if they can be easily removed after the exposure period. This allows more direct comparisons of the results with the chemical effects in humans. The test substance is then removed and the non-toxic, highly fluorescent sodium-fluorescein dye is added to the apical side of the monolayer for 30 minutes. The damage caused by the test substance to the tight junctions is determined by the amount of fluorescein which leaks through the cell layer within a defined period of time.19. The amount of sodium-fluorescein dye that passes through the monolayer and the insert membrane into a set volume of solution present in the well (to which the sodium-fluorescein dye leaks in) is determined by measuring spectrofluorometrically the fluorescein concentration in the well. The amount of fluorescein leakage (FL) is calculated with reference to fluoresence intensity (FI) readings from two controls: a blank control, and a maximum leakage control. The percentage of leakage and therefore amount of damage to the tight junctions is expressed, relative to these controls, for each of the set concentrations of the test substance. Then the FL20 (i.e. concentration that causes 20% FL relative to the value recorded for the untreated confluent monolayer and inserts without cells), is calculated. The FL20 (mg/mL) value is used in the prediction model for identification of ocular corrosives and severe irritants (see paragraph 35).20. Recovery is an important part of a test substance’s toxicity profile that is also assessed by the in vivo ocular irritation test. Preliminary analyses indicated that recovery data (up to 72 h following the chemical exposure) could potentially increase the predictive capacity of INVITTOX Protocol 71 but further evaluation is needed and would benefit from additional data, preferably acquired by further testing (7). This Test Guideline will be updated accordingly as new information and data are considered.PROCEDUREPreparation of the cellular monolayer21. The monolayer of MDCK CB997 cells is prepared using sub-confluent cells growing in cell culture flasks in DMEM/Nutrient Mix F12 (1x concentrate with L-glutamine, 15 mM HEPES, calcium (at a concentration of 1.0-1.8 mM) and 10% heat-inactivated FCS/FBS). Importantly, all media/solutions used throughout the FL assay should contain calcium at a concentration between 1.8 mM (200 mg/L) and 1.0 mM (111 mg/L) to ensure tight junction formation and integrity. Cell passage number range should be controlled to ensure even and4© OECD, (2012)OECD/OCDE 460 reproducible tight junctions formation. Preferably, the cells should be within the passage range 3-30 from thawing because cells within this passage range have similar functionality, which aids assay results to be reproducible.22. Prior to performing the FL test method, the cells are detached from the flask by trypsinisation, centrifuged and an appropriate amount of cells is seeded into the inserts placed in 24-well plates (see Annex I). Twelve mm diameter inserts with membrane of mixed cellulose esters, a thickness of 80-150 µm and a pore size of 0.45 µm, should be used to seed the cells. In the validation study, Millicell-HA 12 mm inserts were used. The properties of the insert and membrane type are important as these may affect cell growth and chemical binding. Certain types of chemicals may bind to the Millicell-HA insert membrane, which could affect the interpretation of results. Proficiency chemicals (see Annex III) should be used to demonstrate equivalency if other membranes are used.23. Chemical binding to the insert membrane is more common for cationic chemicals, such as benzalkonium chloride, which are attracted to the positively charged membrane (8). Chemical binding to the insert membrane may increase the chemical exposure period, leading to an over-estimation of the toxic potential of the chemical, but can also physically reduce the leakage of fluorescein through the insert by binding of the dye to the cationic chemical bound to the insert membrane, leading to an under-estimation of the toxic potential of the chemical. This can be readily monitored by exposing the membrane alone to the top concentration of the chemical tested and then adding sodium-fluorescein dye at the normal concentration for the standard time (no cell control). If binding of the sodium-fluorescein dye occurs, the insert membrane appears yellow after the test material has been washed-off. Thus, it is essential to know the binding properties of the test substance in order to be able to interpret the effect of the chemical on the cells.24. Cell seeding on inserts should produce a confluent monolayer at the time of chemical exposure. 1.6 x 105 cells should be added per insert (400 µL of a cell suspension with a density of 4 x 105 cells / mL). Under these conditions, a confluent monolayer is usually obtained after 96 hours in culture. Inserts should be examined visually prior to seeding, so as to ensure that any damages recorded at the visual control described at paragraph 30 is due to handling.25. The MDCK cell cultures should be kept in incubators in a humidified atmosphere, at 5% ± 1% CO2and 37 ± 1 ºC. The cells should be free of contamination by bacteria, viruses, mycoplasma and fungi.Application of the Test and Control Chemicals26. A fresh stock solution of test substance should be prepared for each experimental run and used within 30 minutes of preparation. Test substances should be prepared in calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS to avoid serum protein binding. Solubility of the chemical at 250 mg/mL in HBSS should be assessed prior to testing. If at this concentration the chemical forms a stable suspension or emulsion (i.e.maintains uniformity and does not settle or separate into more than one phase) over 30 minutes, HBSS can still be used as solvent. However, if the chemical is found to be insoluble in HBSS at this concentration, the use of other test methods instead of FL should be considered. The use of light mineral oil as a solvent, in cases where the chemical is found to be insoluble in HBSS, should be5© OECD, (2012)460OECD/OCDEconsidered with caution as there is not enough data available to conclude on the performance of the FL assay under such conditions.27. All chemicals to be tested are prepared in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS from the stock solution, at five fixed concentrations diluted on a weight per volume basis: 1, 25, 100, 250 mg/mL and a neat or a saturated solution. When testing a solid chemical, a very high concentration of 750 mg/mL should be included. This concentration of chemical may have to be applied on the cells using a positive displacement pipette. If the toxicity is found to be between 25 and 100 mg/mL, the following additional concentrations should be tested twice: 1, 25, 50, 75, 100 mg/mL. The FL20value should be derived from these concentrations provided the acceptance criteria were met.28. The test substances are applied to the confluent cell monolayers after removal of the cell culture medium and washing twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS. Previously, the filters have been visually checked for any pre-existing damages that could be falsely attributed to potential incompatibilities with test chemicals. At least three replicates should be used for each concentration of the test substance and for the controls in each run. After 1 min of exposure at room temperature, the test substance should be carefully removed by aspiration, the monolayer should be washed twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS, and the fluorescein leakage should be immediately measured. 29. Concurrent negative (NC) and positive controls (PC) should be used in each run to demonstrate that monolayer integrity (NC) and sensitivity of the cells (PC) are within a defined historical acceptance range. The suggested PC chemical is Brij 35 (CAS No. 9002-92-0) at 100 mg/mL. This concentration should give approximately 30% fluorescein leakage (acceptable range 20-40% fluorescein leakage, i.e. damage to cell layer). The suggested NC chemical is calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS (untreated, blank control).A maximum leakage control should also be included in each run to allow for the calculation of FL20 values. Maximum leakage is determined using a control insert without cells.Determination of fluorescein permeability30. Immediately after removal of the test and control substances, 400μL of 0.1 mg/mL sodium-fluorescein solution (0.01% (w/v) in calcium-containing [at a concentration of 1.0-1.8 mM], phenol red-free, HBSS) is added to the Millicell-HA inserts. The cultures are kept for 30 minutes at room temperature. At the end of the incubation with fluorescein, the inserts are carefully removed from each well. Visual check is performed on each filter and any damage which may have occurred during handling is recorded.31. The amount of fluorescein that leaked through the monolayer and the insert is quantified in the solution which remained in the wells after removal of the inserts. Measurements are done in a spectrofluorometer at excitation and emission wavelengths of 485 nm and 530 nm, respectively. The sensitivity of the spectrofluorometer should be set so that there is the highest numerical difference between the maximum FL (insert with no cells) and the minimum FL (insert with confluent monolayer treated with NC). Because of the differences in the used spectrofluorometer, it is suggested that a sensitivity is used which will give fluorescence intensity > 4000 at the maximum fluorescein leakage control. The maximum FL value should not be6© OECD, (2012)OECD/OCDE 460 greater than 9999. The maximum fluorescence leakage intensity should fall within the linear range of the spectrofluorometer used.Interpretation of results and Prediction model32. The amount of FL is proportional to the chemical-induced damage to the tight junctions. The percentage of FL for each tested concentration of chemical is calculated from the FL values obtained for the test substance with reference to FL values from the NC (reading from the confluent monolayer of cells treated with the NC) and a maximum leakage control (reading for the amount of FL through an insert without cells).The mean maximum leakage fluorescence intensity = xThe mean 0% leakage fluorescence intensity (NC) = yThe mean 100% leakage is obtained by subtracting the mean 0% leakage from the mean maximum leakage,i.e. x - y = z33. The percentage leakage for each fixed dose is obtained by subtracting the 0% leakage to the mean fluorescence intensity of the three replicate readings (m), and dividing this value by the 100% leakage, i.e. %FL = [(m-y) / z] x 100%, where:m = the mean fluorescence intensity of the three replicate measurements for the concentration involved% FL = the percent of the fluorescein which leaks through the cell layer34. The following equation for the calculation of the chemical concentration causing 20% FL should be applied:FL D = [(A-B) / (C-B)] x (M C –M B) + M BWhere:D = % of inhibitionA = % damage (20% fluorescein leakage)B = % fluorescein leakage < AC = % fluorescein leakage > AM C = Concentration (mg/mL) of CM B = Concentration (mg/mL) of B35. The cut-off value of FL20 for predicting chemicals as ocular corrosives/severe irritants is given below:7© OECD, (2012)460OECD/OCDE36. The FL test method is recommended only for the identification of water soluble ocular corrosives and severe irritants (UN GHS Category 1, EU CLP Category 1, U.S. EPA Category I) (see paragraphs 1 and 10).37. In order to identify water soluble chemicals (substances and mixtures) (4) (7) (8) as "inducing serious eye damage" (UN GHS/EU CLP Category 1) or as an "ocular corrosive or severe irritant" (U.S. EPA Category I), the test substance should induce an FL20 value of ≤ 100 mg/mL.Acceptance of results38. The mean maximum fluorescein leakage value (x) should be higher than 4000 (see paragraph 31), the mean 0% leakage (y) should be equal or lower than 300, and the mean 100% leakage (z) should fall between 3700 and 6000.39. A test is considered acceptable if the positive control produced 20% to 40% damage to the cell layer (measure as % fluorescein leakage).DATA AND REPORTINGData40. For each run, data from individual replicate wells (e.g. fluorescence intensity values and calculated percentage FL data for each test substance, including classification) should be reported in tabular form. In addition, means ± SD of individual replicate measurements in each run should be reported.Test Report41. The test report should include the following information:Test and Control Substances-Chemical name(s) such as the structural name used by the Chemical Abstracts Service (CAS), followed by other names, if known;-Chemical CAS number, if known;-Purity and composition of the substance or mixture (in percentage(s) by weight), to the extent this information is available;-Physical-chemical properties relevant to the conduct of the study (e.g. physical state, volatility, pH, stability, water solubility, chemical class);-Treatment of the test/control substance prior to testing, if applicable (e.g. warming, grinding);-Storage conditions;Justification of the Test Method and Protocol Used-Should include considerations regarding applicability domain and limitations of the test method;Test Conditions8© OECD, (2012)OECD/OCDE 460 -Description of cell system used, including certificate of authenticity and the mycoplasma status of the cell line;-Details of test procedure used;-Test substance concentration(s) used;-Duration of exposure to the test substance;-Duration of incubation with fluorescein;-Description of any modifications of the test procedure;-Description of evaluation criteria used;-Reference to historical data of the model (e.g. negative and positive controls, benchmark chemicals, if applicable);-Information on the technical proficiency demonstrated by the laboratory;Results-Tabulation of data from individual test substances and controls for each run and each replicate measurement (including individual results, means and SDs);-The derived classification(s) with reference to the prediction model and/or decision criteria used;-Description of other effects observed;Discussion of the Results-Should include considerations regarding a non-conclusive outcome (paragraph 35: FL20 > 100 mg/mL) and further testing;Conclusions9© OECD, (2012)460OECD/OCDELITERATURE1.UN (2009), United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Third revised edition, New York & Geneva: United Nations Publications.ISBN: 978-92-1-117006-1. Available at:[/trans/danger/publi/ghs/ghs_rev03/03files_e.html]2.EC (2008), Regulation (EC) No 1272/2008 of the European Parliament and of the Council of16 December 2008 on classification, labelling and packaging of substances and mixtures,amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006, Official Journal of the European Union L353, 1-1355.3.U.S. EPA (1996), Label Review Manual: 2nd Edition, EPA737-B-96-001, Washington DC:U.S. Environmental Protection Agency.4.EC-ECVAM (2009), Statement on the scientific validity of cytotoxicity/cell-function based invitro assays for eye irritation testing. Available under Publications at: [http://ecvam.jrc.it/index.htm]5.Scott, L. et al. (2010), A proposed eye irritation testing strategy to reduce and replace in vivostudies using Bottom-Up and Top-Down approaches, Toxicol. In Vitro 24, 1-9.6.OECD (2002), Test No. 405: Acute Eye Irritation/Corrosion, OECD Guidelines for theTesting of Chemicals, Section 4, OECD Publishing. doi: 10.1787/9789264070646-en7.EC-ECVAM (1999), INVITOX Protocol 71: Fluorescein Leakage Test, Ispra, Italy:European Centre for the Validation of Alternative Methods (ECVAM). Available at: [http://ecvam-dbalm.jrc.ec.europa.eu]8.EC-ECVAM (2008), Fluorescein Leakage Assay Background Review Document as anAlternative Method for Eye Irritation Testing. Available under Validation Study Documents, Section Eye Irritation at: [http://ecvam.jrc.it/index.htm]9.OECD (2005), Guidance Document on the Validation and International Acceptance of Newor Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No. 34. OECD, Paris. Available at: [/env/testguidelines]10© OECD, (2012)。
TAK-779 229005-80-5 GlpBio
Product Data SheetProduct Name:TAK-779Cat. No.:GC37724Chemical PropertiesCas No.229005-80-5ChemicalNameN/ACanonicalSMILESC[N+](C)(CC1=CC=C(NC(C2=CC3=CC(C4=CC=C(C)C=C4)=CC=C3CCC2)=O)C=C1)C5CCOCC5.[Cl-] Formula C33H39ClN2O2M.Wt531.13Solubility Soluble in DMSO Storage Store at -20°CGeneral tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.Shipping Condition Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request.StructureBackgroundTAK-779 is a potent and selective nonpeptide antagonist of CCR5 and CXCR3, with a Ki of 1.1 nM for CCR5, andeffectively and selectively inhibits R5 HIV-1, with EC50 and EC90 of 1.2 nM and 5.7 nM, respectively, in MAGI-CCR5Product Data Sheetcells. MIP-1α-CCR5|1 nM (IC50, in CHO/CCR5 cells)|MIP-1β-CCR5|1 nM (IC50, in CHO/CCR5 cells)|RANTES-CCR5|1.4 nM (IC50, in CHO/CCR5 cells)|MCP-1-CCR2b|27 nM (IC50, in CHO/CCR5 cells)|R5 HIV-1 (Ba-L)|1.2 nM (EC50, in MAGI-CCR5 cells)|R5 HIV-1 (KK)|1.6 nM (EC50, in PBMCs)|R5 HIV-1 (HHA)|3.2 nM (EC50, in PBMCs)|R5 HIV-1 (CTV)|3.5 nM (EC50, in PBMCs)|R5 HIV-1 (Ba-L)|3.7 nM (EC50, in PBMCs)|R5 HIV-1 (Ba-L)|5.7 nM (EC90, in MAGI-CCR5 cells)|R5 HIV-1 (HHA)|7.5 nM (EC90, in PBMCs)|R5 HIV-1 (Ba-L)|12.8 nM (EC90, in PBMCs)|R5 HIV-1 (KK)|20.8 nM (EC90, in PBMCs)|R5 HIV-1 (CTV)|27 nM (EC90, in PBMCs)|mCXCR3|369 nM (IC50, in PBMCs)TAK-779 is a potent and selective nonpeptide antagonist of CCR5, with a Ki of 1.1 nM, and effectively and selectively inhibits R5 HIV-1, with EC50 and EC90 of 1.2 nM and 5.7 nM, respectively, in MAGI-CCR5 cells. TAK-779 less potently blocks the binding of [125I]-monocyte chemotactic protein 1 to CCR2b in CHO/CCR2b cells, with an IC50 for CCR2b of 27 nM. TAK-779 also completely inhibits the binding of [125I]-RANTES to CHO/CCR5 cells with an IC50 of 1.4 nM. TAK-779 (20 nM) selectively inhibits CCR5-mediated Ca2+-signaling. In addition, TAK-779 shows no inhibition on X4 HIV-1 strains[1]. TAK-779 is an antagonist of CXCR3, and inhibits the migration of T cells but not T cellproliferation[2].TAK-779 (10 mg/kg per day, s.c.) significantly prolongs the allograft survival of the rat intestinal transplantation model. TAK-779 also decreases the number of CD4+ as well as CD8+ T cells in spleen, blood and recipient mesenteric lymph nodes (MLN)[2]. TAK-779 (150 µg per mouse, s.c.) supppresses the development of experimental autoimmune encephalomyelitis (EAE) in myelin oligodendrocyte glycoprotein (MOG)-immunizedC57BL/6 mice. TAK-779 decreases the infiltration of CXCR3 and CCR5 bearing leukocytes into the spinal cord.TAK-779 does not alter myelin oligodendrocyte glycoprotein (MOG)-specific immune responses or affect the potential of MOG-specific T cells to transfer experimental autoimmune encephalomyelitis (EAE)[3].[1]. Baba M, et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5698-703. [2]. Takama Y, et al. Effects of a calcineurin inhibitor,FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model. Transpl Immunol. 2011 Jul;25(1):49-55. [3]. Ni J, et al. The chemokine receptor antagonist, TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, withoutaffecting T cell function. Br J Pharmacol. 2009 Dec;158(8):2046-56. [4]. Gao P, et al. The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3. J Leukoc Biol. 2003 Feb;73(2):273-80.。
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Product Name:
TAK-779CAS No.:
229005-80-5Cat. No.:
HY-13406
Product Data Sheet
MWt:
531.13Formula:
C33H39ClN2O2Purity :>98%
Solubility:
DMSO
y Mechanisms:Biological Activity:
TAK 779is a highly potent and selective nonpeptide CCR5antagonist with a IC50value of 14nM in
Pathways:Anti-infection; Target:HIV
Pathways:GPCR/G protein; Target:CCR
Pathways:Immunology/Inflammation; Target:CCR TAK-779 is a highly potent and selective nonpeptide CCR5 antagonist with a IC50 value of 1.4 nM in the binding assay, TAK-779 also inhibited the replication of macrophage (M)-tropic HIV-1 (Ba-L strain) in both MAGI-CCR5 cells and PBMCs with EC50 values of 1.2 and 3.7 nM, respectively.
IC50 value: 1.4 nM [1]
Target: CCR5in vitro: TAK-779, a nonpeptide compound with a small molecular weight (Mr 531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibition of beta-chemokine receptors by TAK-779 appeared to be specific to References:
[1]. Shiraishi M, et al. Discovery of novel, potent, and selective small-molecule CCR5 antagonists as anti-HIV-1 agents: synthesis and biological evaluation of anilide derivatives with a quaternary ammonium moiety. J Med Chem. 2000 May 18;43(10):2049-63.p y pp p CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1,CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R...
y y ()[2]. Baba M, et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective
anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5698-703.[3]. Dragic T, et al. A binding pocket for a small molecule inhibitor of HIV-1 entry within the
transmembrane helices of CCR5. Proc Natl Acad Sci U S A. 2000 May 9;97(10):5639-44.[4]. Takama Y, et al. Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK- 779, in a rat small intestinal transplantation model. Transpl Immunol. 2011 Jul;25(1):49-55....
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。