EXERCISE 37-2 APA documentation in-text citations

390National Instruments • Tel: (800) 433-3488•Fax: (512) 683-9300•***********•C o u n t e r /T i m e r A c c e s s o r i e s a n d C a b l e sD a t a A c q u i s i t i o n a n d S i g n a l C o n d i t i o n i n gAccessory and Cable Selection ProcessStep 1.Select your counter/timer device from Tables 1 and 2.Step ing Tables 1 and 2 as a guide,determine which accessories are appropriate for that device.Select an accessory using Table 3 as reference.Step ing Tables 1 and 2,determine the appropriate cable solution for your selected counter/timer device and accessory.AccessoriesBNC-2121 (See Figure 1)Connector block with BNC and spring terminal connections for easy connection of I/O signals to counter/timer devices.The BNC-2121 offers spring terminals,as well as eight dedicated and six user-defined BNC connectors,which provide access to all I/O signals.This connector block is also a full-featured test accessory that provides pulse-train,trigger,and quadrature encoder signals.For the connections,refer to the BNC-2121 user guide at /manualsBNC-2121......................................................................................................778289-01Dimensions – 26.7 by 11.2 by 5.5 cm (8.0 by 4.4 by 2.2 in.)CA-1000 (See Figure 2)Configurable signal connectivity solution for connecting counter/timers to different types of standard I/O connectors.You can also incorporate switches and LED indicators.You can place the CA-1000 under a laptop PC,on a benchtop,or in a 19 in.rack.CA-1000......................................................................................................See page 351Dimensions – 30.7 by 25.4 by 4.3 cm (12.1 by 10 by 1.7 in.)Counter/Timer Accessories and CablesFigure 2. CA-1000 Configurable Signal Conditioning SolutionFigure 1. BNC-2121 Connector BlockAccessory DescriptionPage BNC-2121BNC connector block with built-in test features390CA-1000Configurable connector accessory 390SCB-68Shielded screw connector block 391TB-2715Front-mount terminal block for PXI-660x391TBX-68DIN-rail connector block 391CB-68LP Low-cost screw connector block 391CB-68LPR Low-cost screw connector block 391Table 1. Accessories and Cables for PXI-6601 and PCI-6602Table 2. Accessories and Cables for PXI-6602 and PXI-6608Table 3. Overview of AccessoriesPCI-6601, PCI-6602AccessoriesCables BNC-2121, CA-1000, SCB-68,–TBX-68, CB-68LP , and CB-68LPR R6868 or SH68-68-D1TB-2715N/APXI-6602, PXI-6608AccessoriesCables BNC-2121, CA-1000, SCB-68,–TBX-68, CB-68LP , and CB-68LPR R6868 or SH68-68-D1TB-2715Connects directly to the deviceCounter/Timer Accessories and CablesSCB-68 Shielded I/O Connector Block (See Figure 3)Shielded I/O connector block for easy connection of I/O signals to the counter/timer devices.T he screw terminals are housed in a metal enclosure for protection from noise bined with a shielded cable,the SCB-68 provides rugged,very low-noise signal termination.T he SCB-68 also includes two general-purpose breadboard areas.SCB-68..............................................................................................................776844-01Dimensions – 19.5 by 15.2 by 4.5 cm (7.7 by 6.0 by 1.8 in)TB-2715 Terminal Block (See Figure 4)With the T B-2715 terminal block for PXI counter/timer devices,you can connect signals directly without additional cables.Screw terminals provide easy connection of I/O signals.The TB-2715 latches to the front of your PXI module with locking screws and provides strain relief.TB-2715............................................................................................................778242-01Dimensions – 8.43 by 10.41 by 2.03 cm (3.32 by 4.1 by 0.8 in.)TBX-68 I/O Connector Block with DIN-Rail Mounting (See Figure 5)Termination accessory with 68 screw terminals for easy connection of field I/O signals to the counter/timer devices.The TBX-68 is mounted in a protective plastic base with hardware for mounting on a standard DIN rail.TBX-68..............................................................................................................777141-01Dimensions – 12.50 by 10.74 cm (4.92 by 4.23 in.)CB-68LP and CB-68LPR I/O Connector Blocks (See Figure 6)Low-cost termination accessories with 68 screw terminals for easy connection of field I/O signals to the counter/timer devices.The connector blocks include standoffs for use on a desktop or mounting in a custom panel.T he CB-68LP has a vertically mounted 68-pin connector.The CB-68LPR has a right-angle mounted connector for use with with the CA-1000.CB-68LP............................................................................................................777145-01Dimensions – 14.35 by 10.74 cm (5.65 by 4.23 in.)CB-68LPR ........................................................................................................777145-02Dimensions – 7.62 by 16.19 cm (3.00 by 6.36 in.)391National Instruments • Tel: (800) 433-3488•Fax: (512) 683-9300•***********•Counter/Timer Accessories and CablesData Acquisition andSignal ConditioningFigure 6. CB-68LP and CB-68LPR I/O Connector BlocksFigure 5. TBX-68 I/O Connector BlockFigure 4. TB-2715 I/O Terminal BlockFigure 3. SCB-68 Shielded I/O Connector Block392National Instruments • Tel: (800) 433-3488•Fax: (512) 683-9300•***********•C o u n t e r /T i m e r A c c e s s o r i e s a n d C a b l e sD a t a A c q u i s i t i o n a n d S i g n a l C o n d i t i o n i n gCablesRTSI Bus Cables (See Figures 7 and 8)Use RT SI bus cables to connect timing and synchronization signals among measurement,vision,motion,and CAN boards for PCI.For systems using long and short boards,order the extended RTSI cable.2 boards ..........................................................................................................776249-023 boards ..........................................................................................................776249-034 boards ..........................................................................................................776249-045 boards ..........................................................................................................776249-05Extended,5 boards ........................................................................................777562-05SH68-68-D1 Shielded Cable (See Figure 9)Shielded 68-conductor cable terminated with two 68-pin female 0.050 series D-type connectors.This cable connects counter/timer devices to accessories.1 m..................................................................................................................183432-012 m..................................................................................................................183432-02R6868 Ribbon I/O Cable (See Figure 10)68-conductor flat ribbon cable terminated with two 68-pin e this cable to connect the NI PCI-6601 to an accessory.For signal integrity with high-frequency signals,use the SH68-68-D1 with the NI 6602 and NI 6608.1 m..................................................................................................................182482-01Custom Connectivity Components68-Pin Custom Cable Connector/Backshell Kit (See Figure 11)68-pin female mating custom cable kit for use in making custom 68-conductor cables.Solder-cup contacts are available for soldering of cable wires to the connector.68-pin custom cable kit ................................................................................776832-01PCB Mounting ConnectorsPrinted circuit board (PCB) connectors for use in building custom accessories that connect to 68-conductor shielded and ribbon cables.Two connectors are available,one for right-angle and one for vertical mounting onto a PCB.68-pin,male,right-angle mounting..............................................................777600-0168-pin,male,vertical mounting....................................................................777601-01Counter/Timer Accessories and CablesFigure 10. R6868 Ribbon I/O CableFigure 9. SH68-68-D1 Shielded CableFigure 8. Extended RTSI Bus CableFigure 7. RTSI Bus CableFigure 11. 68-Pin Custom Cable Kit。

附录II技术文档制造商拟定的技术文档及其摘要（如适用）应以清晰、有条理、易于搜索和明确的方式呈现，尤其应包括本附录中列出的要素。

1、产品描述和特征，包括变体和附件/配件1.1、产品描述和规格a）产品或贸易名字和产品基本描述包括预期用途和预期使用人群b）附录VI Part C提到的制造商分配给器械的UDI-DL基于UDI系统，或产品代码，序列号或其他可参考的标识就会被立马识别c）预期用途包括以下信息：I）检测或测量物质II）功能，例如：筛查、监测、诊断或辅助诊断、预测、伴随诊断III）旨在检测、定义或区分特定疾病、情况和风险因素IV）自动或非自动V）定性、半定量或定量VI）样本类型VII）预期检测人群VIII）预期使用者IX）另外，伴随诊断需要有相关目标人群和相关药品d）检测方法或仪器操作原则的描述e）产品作为医疗器械的理由f）风险等级和分类规则的应用遵循附录VIIIg）组分的描述，和相关组分里活性成分：抗体、抗原、核酸引物（如适用）如适用：h）样本采集、和器械一起提供的运输材料，或推荐使用规格的描述i）对于自动检测的仪器：描述适用的检测性能或专用检测性能j）对于自动检测试剂：描述适用的仪器性能或专用的仪器性能k）描述与器械一起使用的软件l）拟投放市场的器械的各种配置/变体的说明或完整列表m）描述预期与器械联合使用的配件、其他器械和非器械引申：TUV解读，单独提供的配件需要单独的标签、说明书、包装和证书1.2、参考已上市或类似器械a）己上市同类产品或制造商已制造产品的概览b）已在国际市场上销售的类似产品的概览2、制造商提供的信息一套完整的a）器械上的标签或包装（例如单一包装、销售包装、运输包装）的语言应被预想销往的成员国接受b）使用说明书语言应该被预想销往的成员国接受3、设计和制造消息3.1、设计信息用于理解器械设计阶段的信息应包括：a）描述与器械一起提供或推荐一起使用的关键组分如抗体、抗原、酶和核酸引物b）对于仪器：描述主要子系统、分析技术例如操作原则、控制原理、专用电脑硬件或软件c）对于仪器和软件：描述整个系统d）对于软件：描述数据解读技术及算法e）对于预期用于自我测试或近患者测试的器械：描述自我测试或近患者测试的适用性设计方面3.2、制造信息a）生产制造过程信息，例如生产过程、装配、成品测试、成品的包装信息。

达格列净对ApoE -/-小鼠单核巨噬细胞系统及动脉粥样硬化的影响李 宵1,王丹丹1,马 巍2,李 兵1,贾俊栋1,王泰然1,赵季红3摘要 目的:研究达格列净对载脂蛋白E 基因敲除(ApoE -/-)小鼠动脉粥样硬化(AS )斑块的影响,探讨其抗AS 作用的可能机制㊂方法:16只8周龄雄性ApoE -/-小鼠高脂饮食喂养8周形成AS 模型后,随机分为对照组和实验组,每组8只,实验组予达格列净干预,对照组予同等剂量的生理盐水,两组干预4周后取材㊂油红O 染色检测主动脉及主动脉瓣AS 斑块面积比例;流式细胞术分析循环中单核细胞亚群的比例(Ly6G -CD11b +Ly6C hi 和Ly6G -CD11b +Ly6C lo );免疫荧光染色和共聚焦显微镜成像检测斑块内的巨噬细胞和增殖的巨噬细胞(F4/80和Ki67双阳性细胞);酶联免疫吸附法(ELISA )检测血清中总胆固醇(TC )㊁三酰甘油(TG )㊁高密度脂蛋白胆固醇(HDL -C )㊁低密度脂蛋白胆固醇(LDL -C )的水平㊂结果:实验组与对照组比较有降低主动脉及主动脉瓣斑块负荷的作用,降低循环中Ly6C hi 单核细胞比例,降低斑块内增殖巨噬细胞比例,降低LDL -C 水平,且具有升高HDL -C 作用㊂结论:达格列净可减轻ApoE -/-小鼠AS 负荷的作用,可能与调节单核细胞亚群和血脂代谢相关㊂关键词 动脉粥样硬化;达格列净;单核细胞亚群;增殖巨噬细胞;血脂;实验研究d o i :10.12102/j.i s s n .1672-1349.2024.03.012 动脉粥样硬化(atherosclerosis ,AS )是导致心血管疾病的主要病理生理基础,其发病机制有多种学说,包括脂质浸润学说㊁慢性炎症学说等[1],其特征是动脉壁内膜发生明显的慢性炎症反应,而单核巨噬细胞系统参与了炎症反应的过程,在AS 的进程中发挥了重要作用[2]㊂达格列净作为钠-葡萄糖协同转运体2(sodium glucose co -transporters 2,SGLT2)抑制剂,是用于治疗糖尿病相对新的一类抗高血糖药物㊂国外一些临床研究已经证实,达格列净可以降低糖尿病病人心血管疾病的发生率㊂有临床研究也证实,达格列净能够通过降低核苷酸结合寡聚化结构域样受体蛋白-3的活化,减少白细胞介素-1β(interleukin 1β,IL -1β)㊁白细胞介素-18(interleukin 18,IL -18)等炎性因子的产生和释放,发挥抗AS 形成的功效[3],而单核巨噬细胞系统的变化参与了炎症反应会直接影响动脉粥样硬化的进展[4]㊂因此,设想达格列净是否能通过单核巨噬细胞系统发挥抗炎作用,进而影响动脉粥样硬化进程㊂为了验证该假设,本研究通过给载脂蛋白E 基因敲除(ApoE -/-)小鼠喂食高脂饲料构建AS 模型,并观察达格列净对单核巨噬细胞系统及AS 的影响㊂基金项目 邯郸市科学技术研究与发展计划项目(No.21422083111)作者单位 1.邯郸市中心医院(河北邯郸056001),E -mail :lixiao130406@ ;2.华北医疗健康集团峰峰矿区总医院;3.武警特色医学中心/天津市心血管重塑与靶器官损伤重点实验室引用信息 李宵,王丹丹,马巍,等.达格列净对ApoE -/-小鼠单核巨噬细胞系统及动脉粥样硬化的影响[J ].中西医结合心脑血管病杂志,2024,22(3):470-474.1 材料与方法1.1 主要仪器与试剂油红O 购自Sigma 公司,USA ;酶联免疫吸附法(ELISA )试剂盒购自北京艾然生物有限公司;流式细胞仪(Cytomics FC 500,Beckman Coulter ,USA );荧光显微镜(ECLIPSE 80i ,Nikon ,Japan );激光扫描共聚焦显微镜(TCS SP8,Leica ,Germany );冰冻切片机(CM7500,Leica ,Germany );抗CD11b -PE ㊁抗Ly6G -PerCP/Cy5.5和抗Ly6C -FITC 均购自Biolegend 公司,USA ;抗F4/80㊁抗Ki67均购自Abcam 公司,USA ;FITC 标记山羊抗小鼠二抗㊁TRITC 标记山羊抗兔二抗均购自ImmunoReagents 公司,USA ;Mounting medium with DAPI ,购自中杉金桥公司,中国;其余国产试剂均为分析纯;高脂饲料及正常饲料购自江苏省南通特洛菲饲料科技有限公司㊂1.2 实验动物16只8周龄ApoE -/-小鼠,雄性,体质量22~25g ,购自北京维通利华实验动物技术有限公司,许可证号为SCXK (京)2102-0001,实验前均经DNA 检测为纯合子㊂1.3 动物模型的制备㊁标本的采集和处理将小鼠高脂饮食喂养8周形成AS 后,随机分为对照组和实验组,每组8只㊂实验组喂食达格列净剂量为1mg/(kg ㊃d ),对照组给予同等剂量的生理盐水㊂喂养4周后,麻醉并通过摘眼球取血,第1滴血用于检测单核细胞亚群,剩余血液制备血清检测总胆固醇(total cholesterol ,TC )㊁三酰甘油(triglycerides ,TG)㊁高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)㊁低密度脂蛋白胆固醇(low density lipoprotein cholesterol,LDL-C)的水平,留取主动脉及主动脉瓣进行病理染色㊂本实验小鼠的饲养㊁模型制备㊁标本采集及处理均在天津市心血管重塑与靶器官损伤重点实验室进行㊂1.4油红O染色主动脉和主动脉瓣进行油红O染色,在光学显微镜下观察主动脉和主动脉瓣的斑块及斑块负荷㊂具体方法参照本实验室前期研究[5],采用图像分析软件(Image Pro Plus,IPP)计算主动脉斑块的面积㊁主动脉的面积㊁主动脉瓣斑块面积和血管横断面面积,主动脉斑块负荷=主动脉斑块面积/主动脉面积,主动脉瓣斑块负荷=主动脉瓣斑块面积/血管横断面面积㊂1.5流式细胞术检测循环Ly6G-CD11b+Ly6C hi单核细胞亚群比例参照本实验室前期建立的方法进行检测[5],具体步骤:取100μL抗凝全血加入Ly6G-PerCP/Cy5.5, Ly6C-FITC,CD11b-PE,cell staining buffer吹打混匀,室温避光孵育15min,加入600μL红细胞裂解液,上机检测,FlowJo7.6.1软件分析流式细胞术结果㊂1.6免疫荧光染色参照本实验室前期建立的方法进行免疫荧光染色[5],将冷冻的主动脉瓣切片于室温解冻,1%的TritonX-100破膜20min,山羊血清封闭20min,加入大鼠抗小鼠单抗F4/80(1ʒ100)和兔抗小鼠多抗Ki67 (1ʒ300)4ħ孵育过夜,次日加入山羊抗兔IgG, TRITC和山羊抗大鼠Ig G,FITC,室温孵育1h,含DAPI的防淬灭封片剂封片㊂巨噬细胞和增殖巨噬细胞密度计算如下,采用图像分析软件IPP计算荧光显微镜拍摄视野内巨噬细胞和增殖巨噬细胞(F4/80和Ki67双阳性细胞)个数和斑块面积,增殖巨噬细胞比例=增殖巨噬细胞密度/巨噬细胞密度㊂1.7血清血脂水平测定按照本实验室前期建立的方法[5],取出血清复温后,按照试剂盒说明书进行实验操作,ELISA分析TC㊁TG㊁LDL-C㊁HDL-C水平㊂1.8统计学处理采用Graph Pad Prism6进行统计学分析,符合正态分布的定量资料以均数ʃ标准差(xʃs)表示,组间比较采取独立样本t检验,以P<0.05为差异有统计学意义㊂2结果2.1各组油红O染色结果主动脉经油红O染色,红色即为AS斑块区域(见图1),实验组与对照组比较,主动脉内斑块负荷量减轻,差异有统计学意义(0.150ʃ0.007与0.121ʃ0.007,P=0.010)㊂主动脉瓣经油红O染色,红色即为AS斑块区域(见图2);经统计学分析后提示,实验组主动脉瓣的斑块负荷较对照组降低,差异有统计学意义(0.3299ʃ0.0090与0.2700ʃ0.0125,P=0.0016)㊂详见图3㊂图1主动脉油红O 染色图2主动脉瓣油红O染色(ˑ10)图3油红O染色结果分析(A为两组主动脉斑块负荷比较;B为两组主动脉瓣斑负荷比较㊂两组比较,*P<0.05,#P<0.01)2.2循环中单核细胞亚群比值结果循环中单核细胞亚群的设门策略详见图4,通过FlowJo7.6.1软件分析数据,实验组与对照组比较,循环中Ly6C hi比例下降,差异有统计学意义(0.3299ʃ0.0090与0.2700ʃ0.0125,P=0.0016)㊂详见图5㊂图4流式细胞术检测循环中单核细胞亚群的设门策略图5单核细胞亚群比例结果(两组比较,*P<0.01)2.3主动脉瓣AS斑块内增殖巨噬细胞比例主动脉瓣经免疫荧光染色后,通过激光共聚焦观察,其中蓝色为DAPI标记细胞核所激发荧光,绿色为FITC标记F4/80所激发荧光,红色为TRITC标记Ki67所激发荧光,结果所示DAPI和FITC双阳性表示巨噬细胞(蓝色和绿色共存),DAPI㊁FITC和TRITC三色标记同时在一个细胞则证明为增殖的巨噬细胞(粉色和绿色共存,见图6)㊂实验组与对照组比较,斑块内增殖巨噬细胞比例下降,差异有统计学意义(0.3500ʃ0.0224与0.2738ʃ0.0246,P=0.0379)㊂详见图7㊂图6激光扫描共聚焦显微镜检测主动脉瓣增殖巨噬细胞(绿色为F4/80,红色为Ki67,蓝色为细胞核)图7主动脉瓣内增殖巨噬细胞占巨噬细胞百分比(两组比较,*P<0.01)2.4小鼠血清中血脂比较实验组与对照组比较,血清中TG水平下降,差异有统计学意义[(3.61ʃ0.17)mmol/L与(0.29ʃ0.22)mmol/L,P=0.021],HDL-C的水平升高,差异有统计学意义[(2.72ʃ0.16)mmol/L与(3.15ʃ0.13)mmol/L,P=0.048],但TC[(24.06ʃ0.99)mmol/L与(23.16ʃ0.95)mmol/L,P=0.525]及LDL-C[(22.68ʃ0.79)mmol/L与(21.10ʃ1.11)mmol/L,P=0.262]水平下降,两组比较差异无统计学意义㊂详见图8㊂图8两组血清中血脂水平比较(n=8)(A为TC水平;B为TG水平;C为HDL-C水平;D为LDL-C水平㊂两组比较,*P<0.05)3讨论AS是心血管疾病㊁外周动脉及脑血管疾病发病的基础,动脉血管内皮下斑块的形成导致血管腔明显狭窄,引起组织缺血缺氧,大部分心肌梗死是由于AS斑块破裂导致自发血栓形成阻塞血管引起的,这也是全球最常见的死因[6]㊂AS的药物治疗非常有限主要是抗血小板聚集㊁调脂稳斑㊁降低心肌耗氧量㊁抑制心室重塑,近年来,有研究发现,达格列净可以降低大鼠心肌组织中的肿瘤坏死因子-α(TNF-α)㊁白细胞介素(IL)-6㊁IL-1β㊁单核细胞趋化因子-1(MCP-1)的水平,延缓心室重塑进程[7];也有研究发现,达格列净可通过抑制炎性因子IL-1β㊁IL-18的产生和释放,降低糖尿病ApoE-/-小鼠主动脉及主动脉瓣区AS斑块负荷[3], 2021年‘老年人慢性心力衰竭诊治中国专家共识(2021)“中推荐SGLT-2抑制剂可有效降低心力衰竭病人死亡率,适用于美国纽约心脏病协会(NYHA)心功能分级Ⅱ~Ⅳ级射血分数降低的心力衰竭(HFrEF)病人[8],达格列净对心血管保护作用的证据越来越多㊂本研究发现,达格列净可以降低主动脉及主动脉瓣斑块的负荷,存在抑制AS进程的作用㊂单核巨噬细胞系统可以影响AS的进程,小鼠循环中单核细胞通过Ly6C的表达量不同可分为Ly6C hi和Ly6C lo两种亚型,发挥不同的功能,健康小鼠的单核细胞中50%~ 60%为Ly6C hi亚型,机体发生炎症反应的状态下,骨髓和脾脏中单核细胞的生成增加,使Ly6C hi单核细胞亚型明显升高,发挥抗炎作用[9]㊂通过单核细胞亚群结果的柱状图,发现达格列净可降低Ly6C hi亚型的比例,抑制炎症反应㊂传统的观点认为单核细胞通过血液循环到达局部组织分化为巨噬细胞,但是近些年有研究证实了巨噬细胞在局部有增殖的现象,并且增殖巨噬细胞在动脉粥样硬化斑块进展过程中发挥了重要作用[10],具体机制可能是平滑肌细胞和内皮细胞分泌的巨噬细胞集落刺激因子刺激局部巨噬细胞的自我增殖[11]㊂本课题组前期研究通过免疫荧光染色技术证明了斑块内增殖巨噬细胞的存在[12],提示达格列净存在降低主动脉瓣斑块内增殖巨噬细胞比例的作用,存在抑制AS的进程㊂由此分析,达格列净可能通过调节单核巨噬细胞系统发挥抑制AS进展的作用㊂脂质代谢紊乱是AS的主要危险因素,目前文献报道SGLT2抑制剂对血脂谱的影响尚无定论,但多为改善或中性结局,也有研究表明可升高LDL-C水平,但具体机制目前尚未明确[13-14]㊂本研究发现,在达格列净有降低TG和升高HDL-C水平的作用,说明达格列净有调节血脂代谢的作用,这也可能是其发挥抗AS 作用的潜在机制㊂综上所述,达格列净可能通过影响单核巨噬细胞系统及血脂代谢发挥抗AS的作用,为AS的治疗提供了新的思路,但具体的分子机制仍需进一步探索㊂参考文献:[1]PEDRO-BOTET J,CLIMENT E,BENAIGES D.Atherosclerosis andinflammation.New therapeutic approaches[J].Med Clin,2020,155(6):256-262.[2]LASZLO G,KEATING SAMUEL T,JOOSTEN LEO A B,et al.Monocyte and macrophage immunometabolism in atherosclerosis[J].Seminars in Immunopathology,2018,40(2):203-214.[3]LENG W L,OUYANG X S,LEI X T,et al.The SGLT-2inhibitordapagliflozin has a therapeutic effect on atherosclerosis indiabetic ApoE-/-mice[J].Mediators of Inflammation,2016,2016:6305735.[4]LIU J X,LI X,JI W J,et al.The dynamics of circulating monocytesubsets and intra-plaque proliferating macrophages during thedevelopment of atherosclerosis in ApoE-/-mice[J].InternationalHeart Journal,2019,60(3):746-755.[5]李宵,周欣,姬文婕,等.载脂蛋白E基因敲除小鼠动脉粥样硬化病变进展过程中循环单核细胞亚群和斑块内增殖巨噬细胞的动态变化[J].中国动脉硬化杂志,2015,23(1):24-28.[6]WOLF D,LEY K.Immunity and inflammation in atherosclerosis[J].Circulation Research,2019,124(2):315-327.[7]LEE T M,CHANG N C,LIN S Z.Dapagliflozin,a selective SGLT2Inhibitor,attenuated cardiac fibrosis by regulating themacrophage polarization via STAT3signaling in infarcted rathearts[J].Free Radical Biology&Medicine,2017,104:298-310.[8]中华医学会老年医学分会心血管疾病学组,‘老年慢性心力衰竭诊治中国专家共识“编写组.老年人慢性心力衰竭诊治中国专家共识(2021)[J].中华老年医学杂志,2021,40(5):550-561. [9]DENNEY L,KOK W L,COLE S L,et al.Activation of invariant NKTcells in early phase of experimental autoimmune encephalomyelitisresults in differentiation of Ly6C hi inflammatory monocyte to M2macrophages and improved outcome[J].Journal of Immunology,2012,189(2):551-557.[10]ROBBINS C S,HILGENDORF I,WEBER G F,et al.Localproliferation dominates lesional macrophage accumulation inatherosclerosis[J].Nature Medicine,2013,19(9):1166-1172. [11]SINHA S K,MIIKEDA A,FOULADIAN Z,et al.Local M-CSF(macrophage colony-stimulating factor)expression regulatesmacrophage proliferation and apoptosis in atherosclerosis[J].Arteriosclerosis,Thrombosis,and Vascular Biology,2021,41(1):220-233.[12]LI T,WANG W,LI Y X,et al.Pseudolaric acid B attenuatesatherosclerosis progression and inflammation by suppressingPPARγ-mediated NF-κB activation[J].International Immunopharmacology,2018,59:76-85.[13]SA-NGUANMOO P,TANAJAK P,KERDPHOO S,et al.SGLT2-inhibitor and DPP-4inhibitor improve brain function viaattenuating mitochondrial dysfunction,insulin resistance,inflammation,and apoptosis in HFD-induced obese rats[J].Toxicology and Applied Pharmacology,2017,333:43-50. [14]CHA S A,PARK Y M,YUN J S,et al.A comparison of effects ofDPP-4inhibitor and SGLT2inhibitor on lipid profile in patientswith type2diabetes[J].Lipids in Health and Disease,2017,16(1):58.(收稿日期:2022-04-12)(本文编辑王雅洁)。

Volume 25 Number 27EP7-A2 ISBN 1-56238-584-4 ISSN 0273-3099Interference Testing in Clinical Chemistry; Approved Guideline— Second EditionRobert J. McEnroe, PhD Mary F. Burritt, PhD Donald M. Powers, PhD Douglas W. Rheinheimer, MT Brian H. Wallace, PhDAbstractClinical and Laboratory Standards Institute document EP7-A2—Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition is intended to promote uniformity in the evaluation of interference characteristics of clinical laboratory measurement procedures. EP7 describes procedures for manufacturers to screen potentially interfering substances, to quantify interference effects, and to confirm interference in patient samples. This document also describes procedures for clinical laboratories to verify interference claims, and to investigate discrepant results caused by unsuspected interfering substances. Detailed examples are given. EP7 also contains background information on interference testing concepts, tables of recommended test concentrations for analytes and potential interference, and data collection and analysis worksheets. Clinical and Laboratory Standards Institute (CLSI). Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition. CLSI document EP7-A2 (ISBN 1-56238-584-4). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2005.The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail:customerservice@; Website: Volume 25EP7-A2ContentsAbstract ....................................................................................................................................................i Committee Membership........................................................................................................................ iii Foreword .............................................................................................................................................. vii 1 2 Scope..........................................................................................................................................1 Introduction................................................................................................................................1 2.1 2.2 3 4 5 Measurement Procedures ..............................................................................................1 Concepts and Scientific Principles................................................................................2Standard Precautions..................................................................................................................4 Definitions .................................................................................................................................5 Decision Criteria for Interference Testing .................................................................................8 5.1 5.2 5.3 5.4 5.5 Clinical Acceptability Criteria ......................................................................................9 Statistical Significance and Power................................................................................9 Analyte Test Concentrations.......................................................................................10 Potential Interfering Substances .................................................................................10 Interferent Test Concentrations...................................................................................11 Training and Familiarization.......................................................................................12 Precision Verification .................................................................................................12 Trueness Verification..................................................................................................12 Carryover Assessment ................................................................................................12 Quality Control ...........................................................................................................12 Safety and Waste Disposal..........................................................................................13 Interference Screen .....................................................................................................13 Characterization of Interference Effects .....................................................................20 Evaluating Combinations of Analyte and Interferent(s) .............................................26 Experimental Design...................................................................................................26 Comparative Measurement Procedure........................................................................27 Patient Populations .....................................................................................................27 Experimental Procedure..............................................................................................28 Data Analysis..............................................................................................................28 Interpretation of Results..............................................................................................31 Establishing Interference Claims ................................................................................32 Verifying Analytical Specificity .................................................................................34 Validating Analytical Specificity................................................................................35 Verifying Interference and Specificity Claims ...........................................................366Quality Assurance and Safety ..................................................................................................12 6.1 6.2 6.3 6.4 6.5 6.67Estimation of Interference Characteristics...............................................................................13 7.1 7.2 7.38Evaluating Interference Using Patient Specimens ...................................................................26 8.1 8.2 8.3 8.4 8.5 8.69Establishing, Validating, and Verifying Interference Claims ..................................................31 9.1 9.2 9.3 9.4vNumber 27EP7-A2Contents (Continued)10 Investigating Discrepant Patient Results..................................................................................36 10.1 10.2 10.3 10.4 10.5 10.6 Verify System Performance........................................................................................37 Evaluate Sample Quality ............................................................................................37 Confirm the Original Result........................................................................................37 Identify Potentially Interfering Substances.................................................................38 Determine the Probable Interferent.............................................................................38 Characterize the Interference ......................................................................................39References.............................................................................................................................................40 Appendix A. Guidelines for Specific Measurement Procedures .........................................................43 Appendix B. Analyte Test Concentrations ..........................................................................................46 Appendix C. Interferent Test Concentrations ......................................................................................49 Appendix D. Interference Test Concentrations for Endogenous Analytes ..........................................75 Appendix E. Worksheets .....................................................................................................................79 Appendix F. Calculation of Replicates for Dose-Response Tests .......................................................87 Appendix G. Preparation of Test Solutions for Interference Testing ..................................................90 Summary of Consensus Comments and Committee Responses ...........................................................96 Summary of Consensus/Delegate Comments and Committee Responses..........................................101 The Quality System Approach............................................................................................................104 Related CLSI/NCCLS Publications ....................................................................................................105viVolume 25EP7-A2ForewordInterfering substances can be a significant source of error in clinical laboratory measurements.1-3 Such errors may, in some cases, represent a hazard to the patient. While precision is routinely monitored by internal quality control, and accuracy can be verified by comparison to reference materials or procedures, laboratories cannot easily detect error caused by interfering substances. Therefore, manufacturers of in vitro diagnostic (IVD) analytical systems must include evaluation of the effects of the potentially interfering substances in their risk analyses at the design stage. Although continuously improving the specificity of measurement procedures is a desirable goal, compromise is sometimes necessary to meet the needs of clinical laboratories. The purpose of this document is to enable manufacturers and laboratories to evaluate interfering substances in the context of medical needs and to inform their customers of known sources of medically significant error. This guideline identifies potential hazards to be evaluated in the risk management process described in ISO 14971.4 To accommodate the variety of existing and future measurement procedures, we provided guidance instead of rigid protocols. The subcommittee struck a balance between consistency of structured protocols and flexibility to accommodate the technology being evaluated. Laboratorians and manufacturers need to understand the scientific concepts, make informed choices, and work together toward the common goal of improving patient care. Clearly, identifying an interference effect, evaluating its medical significance, determining its underlying cause, and ultimately improving the measurement procedure requires close cooperation between laboratory and manufacturer. Background information is included to explain key chemical and statistical concepts. Please note that this document focuses on interference with analytical processes. It does not address physiological effects caused by drugs and their metabolites. The IFCC has issued a series of recommendations on drug effects5-7 that have been published as a compendium.8 Comprehensive literature surveys of the analytical and physiological effects of drugs and other substances have been published.9-11 The basic substance of EP7-A2 remains unchanged. A thorough review of the exogenous and endogenous compounds recommended for testing was performed. Each drug or drug metabolite was systematically categorized into specific drug classes. This guideline was developed to inform the reader and provide a logical approach to complete the evaluation of the effects of potentially interfering compounds on the measurement procedure test results. The guideline is intended to make the decision easier by basing it on reasonable, objective criteria. We now ask the reader to give us comments and suggestions. Each comment and suggestion will be considered carefully at the next revision.A Note on TerminologyCLSI, as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, ISO, and CEN documents; and that legally required use of terms, regional usage, and different consensus timelines are all obstacles to harmonization. Despite these obstacles, CLSI recognizes that harmonization of terms facilitates the global application of standards and is an area that needs immediate attention. Implementation of this policy must be an evolutionary and educational process that begins with new projects and revisions of existing documents. In order to align the usage of terminology in this document with that of ISO, the following terms are used in EP7-A2: viiNumber 27EP7-A2The term trueness has replaced the term accuracy when referring to the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. Accuracy, in its metrological sense, refers to the closeness of the agreement between the result of a single measurement and a true value of a measurand, thus comprising both random and systematic effects. The term measurement procedure has replaced the terms method, analytical method, and analytical system for a set of operations used in the performance of particular measurements according to a given method. The term assay has been replaced by method, measurement procedure, measurement, analyze, and analysis as appropriate. At this time, due to user unfamiliarity, the term examination is not used in this edition of EP7. The terms specimen and sample are both used in this document, with specimen reserved for material collected directly from the patient, and sample reserved for aliquots of the patient specimen and for processed materials (e.g., PT samples, reference materials). The term analyte is used appropriately in this document. The term analyte is used to represent the particular component of interest to the patient diagnosis, while the term measurand is used to describe the specific quantity that is measured by a particular measurement procedure (i.e., the measurand describes what is actually causing the result of the measurement). This important difference can be subtle, since it can be due to the detection of different measurands in the procedures being compared. The term precision is a measure of “closeness of agreement between independent test/measurement results obtained under stipulated conditions.”12 The terms in this document are consistent with uses defined in the ISO 3534 and ISO 5725 series of standards. At this time, due to user unfamiliarity and for the sake of the practicability of the guideline, it is important to point out that the working group has chosen not to replace the term interfering substance or interferent with the VIM (International Vocabulary of Basic and General Terms in Metrology) term influence quantity (i.e., quantity that is not the measurand but that affects the result of the measurement). The users of EP7 should understand that the fundamental meanings of the terms are identical, and to facilitate understanding, the terms are defined along with their ISO counterparts in the guideline’s Definitions section. All terms and definitions will be reviewed again for consistency with international use, and revised appropriately during the next scheduled revision of this document.Key WordsEvaluation, hazard analysis, interference, interferent, matrix effects, performance claims, risk management, specificity, validation, verificationviiiVolume 25EP7-A2Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition1 ScopeThis document is intended to serve two purposes: 1) to assist manufacturers and other developers of laboratory measurement procedures in characterizing the susceptibility of measurement procedures to interfering substances, by offering scientifically valid experimental designs, by specifying the relevant substances and concentrations to be tested, and by clarifying appropriate data analysis and interpretation, so that potential hazards can be evaluated and meaningful interference claims may be provided to users; and 2) to assist clinical laboratories in investigating discrepant results due to interfering substances, by defining a systematic investigation strategy, by specifying data collection and analysis requirements, and by promoting greater cooperation between laboratory users and manufacturers, so that new interferences can be identified, disclosed, and ultimately eliminated. This guideline is intended for manufacturers of in vitro diagnostic medical devices and clinical laboratories. Manufacturers and other developers of laboratory measurement procedures are responsible for characterizing the analytical performance of their procedures and analyzing hazards to patients caused by errors due to interfering substances. Manufacturers are required to provide information about interference susceptibility to those who use their systems. NOTE: The term “manufacturer,” for the purpose of this document, is used to mean anyone that develops a measurement procedure for use in a clinical laboratory. Clinical laboratories are responsible for ensuring that measurement procedures are specific enough to meet the needs of their physician clients. Laboratories should also investigate discrepant results, identify interfering substances, and provide objective feedback to the manufacturers who supply their analysis systems.22.1IntroductionMeasurement ProceduresAny measurement procedure, quantitative or qualitative, may be subject to interference. This document is written for a broad spectrum of measurement procedures and analyzers. Modification may be necessary to accommodate the particular characteristics of the procedure being evaluated. Two specific method principles (i.e., separation techniques and immunochemical measurement procedures) are discussed in Appendix A. 2.1.1 Specimen TypeInterferences with measurement procedures that use serum, plasma, whole blood, cerebrospinal fluid, urine, and most other body fluids may be evaluated using this guideline. 2.1.2 Interfering SubstancesPotentially interfering substances may originate from the following endogenous and exogenous sources:©Clinical and Laboratory Standards Institute. All rights reserved.1Volume 25EP7-A2Related CLSI/NCCLS Publications*C3-P4 Preparation and Testing of Reagent Water in the Clinical Laboratory; Proposed Guideline—Fourth Edition (2005). This document provides guidance on water purified for clinical laboratory use; methods for monitoring water quality and testing for specific contaminants; and water system design considerations. Statistical Quality Control for Quantitative Measurements: Principles and Definitions; Approved Guideline—Second Edition (1999). This guideline provides definitions of analytical intervals, planning of quality control procedures, and guidance for quality control applications. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Second Edition (2004). This document provides guidance for designing an experiment to precision performance of quantitative measurement methods; recommendations on comparing precision estimates with manufacturers’ precision performance claims and determining comparisons are valid; as well as manufacturers’ guidelines for establishing claims. Guideline— evaluate the the resulting when suchC24-A2EP5-A2EP9-A2Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Second Edition (2002). This document addresses procedures for determining the bias between two clinical methods, and the design of a method comparison experiment using split patient samples and data analysis. Evaluation of Matrix Effects; Approved Guideline—Second Edition (2005). This document provides guidance for evaluating the bias in analyte measurements that is due to the sample matrix (physiological or artificial) when two measurement procedures are compared. A Quality Management System Model for Health Care; Approved Guideline—Second Edition (2004). This document provides a model for providers of healthcare services that will assist with implementation and maintenance of effective quality management systems.EP14-A2HS1-A2Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process; therefore, readers should refer to the most recent editions.©*Clinical and Laboratory Standards Institute. All rights reserved.105。

D2-40D2-40: An Overview of a Unique Immunohistochemical MarkerIntroductionImmunohistochemistry (IHC) is a widely used technique in the field of pathology that allows for the visualization of specific target antigens within tissues. One such marker is D2-40, which has gained significant attention due to its unique properties and potential diagnostic implications. In this article, we will discuss the characteristics of D2-40, its applications, and its relevance in various pathological conditions.D2-40: A Brief BackgroundD2-40, also known as Podoplanin, is a protein encoded by the PDPN gene. Initially identified as a podocyte membrane antigen, it was shown to play a crucial role in the development of lymphatic vessels. D2-40 is characterized by its expression in endothelial cells of lymphatic vessels, certain subsets of macrophages, and some neoplastic cells of mesothelial and epithelial origin.Expression Patterns and LocalizationD2-40 has shown a consistent pattern of expression in lymphatic endothelial cells throughout various organs and tissues. By utilizing D2-40 immunohistochemistry, these lymphatic vessels can be easily identified and distinguished from blood vessels, which are negative for D2-40 expression. The use of D2-40 has proven to be particularly helpful in studying lymphatic metastasis and lymphatic invasion in various cancers, such as breast, lung, and colorectal carcinomas.Applications in Tumor Diagnosis and PrognosisOne of the most significant clinical applications of D2-40 is in tumor diagnosis and prognosis. Research has shown that D2-40 expression is associated with lymphatic vessel density and lymph node metastasis in several types of cancers. In breast cancer, for example, the presence of D2-40-positive tumor cells in the lymphatic vessels has been correlated with an increased risk of nodal metastasis. Similarly, in lung cancer, D2-40 expression has been associated with an unfavorable prognosis.D2-40 as a Diagnostic MarkerThe distinctive expression pattern of D2-40 has made it a valuable diagnostic marker, especially in tumors where lymphatic invasion is an important factor. In malignant mesothelioma, D2-40 has been shown to be highly sensitive and specific for differentiating it from reactive mesothelial hyperplasia. The absence of D2-40 staining in reactive mesothelial cells helps in ruling out the possibility of malignancy, making it a useful tool for accurate diagnosis.Distinctive Features of D2-40Apart from its diagnostic significance, D2-40 also possesses some unique features that make it an interesting marker to study. Research has shown that D2-40 is involved in the regulation of cell migration, invasion, and metastasis. It has been demonstrated that D2-40 interacts with the C-type lectin-like receptor 2 (CLEC-2), leading to platelet aggregation and lymphatic vessel remodeling. These findings suggest potential therapeutic implications for targeting D2-40 in metastatic disease.ConclusionD2-40, or Podoplanin, is a unique immunohistochemical marker with specific expression in lymphatic endothelial cells and some neoplastic cells. It has become an important diagnostic tool in various cancers, aiding in the identification of lymphatic invasion and predicting prognosis. Additionally, its distinct features and involvement in cellular processes make D2-40 an intriguing marker with potential therapeutic applications. Further research and studies are needed to fully explore the role of D2-40 in various pathological conditions and its potential as a therapeutic target.。

Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSDXing-Yun Chai1, Song-Lin Li2, Ping Li1*1Key Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China2Institute of Nanjing Military Command for Drug Control, Nanjing, 210002, People’s Republic of China*Corresponding author: Ping LiKey Laboratory of Modern Chinese Medicines and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210009, People’s Republic of China.E-mail address: lipingli@Tel.: +86-25-8324-2299; 8539-1244; 135********Fax: +86-25-8532-2747AbstractA new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidinB (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb.Simultaneous separation of these seven saponins was achieved on a C18 analytical column with a mixed mobile phase consisting of acetonitrile(A)-water(B)(29:71 v/v) acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min on linear gradient, and then keep isocratic elution with 54%B from 25 to 30min.The drift tube temperature of ELSD was set at 106℃, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0% respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.Keywords: HPLC-ELSD; Flos Lonicerae; Saponins; Quantification1. IntroductionFlos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca,L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae,owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on[4-5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae[6].On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7-11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicera. As a series studies on the comprehensive evaluation of Flos Lonicera, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e.macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) (Fig. 1).2. Experimental2.1. Samples, chemicals and reagentsFive samples of Lonicera species,L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confuse from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06) respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. The voucher specimens were deposited in the department of Pharmacognosy, China Pharmaceutical University, Nanjing, China. Seven saponin reference compounds: macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7) were isolated previously from the dried buds of L. confusa by repeated silica gel, sephadex LH-20 and Rp-18 silica gel column chromatography, their structures were elucidated by comparison of their spectral data (UV, IR, MS, 1H- NMR and 13C-NMR) with references [13-15]. The purity of these saponins were determined to be more than 98% by normalization of the peak areas detected by HPLC with ELSD, and showed very stable in methanol solution.HPLC-grade acetonitrile from Merck (Darmstadt, Germany), the deionized water from Robust (Guangzhou, China), were purchased. The other solvents, purchased from Nanjing Chemical Factory (Nanjing, China) were of analytical grade.2.2. Apparatus and chromatographic conditionsAglient1100 series HPLC apparatus was used. Chromatography was carried out on an Aglient Zorbax SB-C18 column（250 4.6mm, 5.0µm）at a column temperature of 25℃.A Rheodyne 7125i sampling valve (Cotati, USA) equipped with a sample loop of 20µl was used for sample injection. The analog signal from Alltech ELSD 2000 (Alltech, Deerfield, IL, USA)was transmitted to a HP Chemstation for processing through an Agilent 35900E (Agilent Technologies, USA).The optimum resolution was obtained by using a linear gradient elution. The mobile phase was composed of acetonitrile(A) and water(B) which acidified with 0.5% acetic acid. The elution was operated from keeping 29%A for 10min, then gradually to 54%B from 10 to 25 min in linear gradient, and back to the isocratic elution of 54%B from 25 to 30 min.The drift tube temperature for ELSD was set at 106℃and the nitrogen flow-rate was of 2.6 l/min. The chromatographic peaks were identified by comparing their retention time with that of each reference compound tried under the same chromatographic conditions with a series of mobile phases. In addition, spiking samples with the reference compounds further confirmed the identities of the peaks.2.3. Calibration curvesMethanol stock solutions containing seven analytes were prepared and diluted to appropriate concentration for the construction of calibration curves. Six concentrationof the seven analytes’ solution were injected in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were demonstrated in Table1.2.4. Limits of detection and quantificationMethanol stock solution containing seven reference compounds were diluted to a series of appropriate concentrations with methanol, and an aliquot of the diluted solutions were injected into HPLC for analysis.The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD and LOQ for each compound were shown in Table1.2.5. Precision and accuracyIntra- and inter-day variations were chosen to determine the precision of the developed assay. Approximate 2.0g of the pulverized samples of L. macranthoides were weighted, extracted and analyzed as described in 2.6 Sample preparation section. For intra-day variability test, the samples were analyzed in triplicate for three times within one day, while for inter-day variability test, the samples were examined in triplicate for consecutive three days. Variations were expressed by the relative standard deviations. The results were given in Table 2.Recovery test was used to evaluate the accuracy of this method. Accurate amounts of seven saponins were added to approximate 1.0g of L. macranthoides,and then extracted and analyzed as described in 2.6 Sample preparation section. The average recoveries were counted by the formula: recovery (%) = (amount found –original amount)/ amount spiked ×100%, and RSD (%) = (SD/mean) ×100%. The results were given in Table 3.2.6. Sample preparationSamples of Flos Lonicerae were dried at 50℃until constant weight. Approximate 2.0g of the pulverized samples, accurately weighed, was extracted with 60% ethanol in a flask for 4h. The ethanol was evaporated to dryness with a rotary evaporator. Residue was dissolved in water, followed by defatting with 60ml of petroleum ether for 2 times, and then the water solution was evaporated, residue was dissolved with methanol into a 25ml flask. One ml of the methanol solution was drawn and transferred to a 5ml flask, diluted to the mark with methanol. The resultant solution was at last filtrated through a 0.45µm syringe filter (Type Millex-HA, Millipore, USA) and 20µl of the filtrate was injected to HPLC system. The contents of the analytes were determined from the corresponding calibration curves.3. Results and discussionsThe temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In ourprevious work [12], the temperature of drift tube was optimized at 90°C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95°C to 110°C, the flow-rate from 2.2 to 3.0 l/min. Dipsacoside B was selected as the testing saponin for optimizing ELSD conditions, as it was contained in all samples. Eventually, the drift tube temperature of 106℃and a gas flow of 2.6 l/min were optimized to detect the analytes. And these two exact experimental parameters should be strictly controlled in the analytical procedure [16].All calibration curves showed good linear regression (r2 0.9922) within test ranges. Validation studies of this method proved that this assay has good reproducibility. As shown in Table 2, the overall intra- and inter-day variations are less than 6% for all seven analytes. As demonstrated in Table 3, the developed analytical method has good accuracy with the overall recovery of high than 96% for the analytes concerned. The limit of detection (S/N=3) and the limit of quantification (S/N=10) are less than 0.26μg and 0.88μg respectively (Table1), indicating that this HPLC-ELSD method is precise, accurate and se nsitive enough for the quantitative evaluation of major non- chromaphoric saponins in Flos Lonicerae.It has been reported that there are two major types of saponins in Flos Lonicerae, i.e. saponins with hederagenin as aglycone and saponins with oleanolic acid as the aglycone [17]. But hederagenin type saponins of the herb were reported to have distinct activities of liver protection and anti-inflammatory [7-11]. So we adoptedseven hederagenin type saponins as representative markers to establish a quality control method.The newly established HPLC-ELSD method was applied to analyze seven analytes in five plant sources of Flos Lonicerae, i.e. L. japonica,L. hypoglauca,L. confusa,L. similes and L. macranthoides(Table 4). It was found that there were remarkable differences of seven saponins contents between different plant sources of Flos Lonicerae. All seven saponins analyzed could be detected in L. confusa and L. hypoglauca, while only dipsacoside B was detected in L. japonica. Among all seven saponins interested, only dipsacoside B was found in all five plant species of Flos Lonicerae analyzed, and this compound was determined as the major saponin with content of 53.7 mg/g in L. hypoglauca. On the other hand, macranthoidin B was found to be the major saponin with the content higher than 41.0mg/g in L. macranthoides,L. confusa, and L. similis, while the contents of other analytes were much lower.In our previous study [12], overall HPLC profiles of iridoid glucosides was used to qualitatively and quantitatively distinguish different origins of Flos Lonicerae. As shown in Fig.2, the chromatogram profiles of L. confusa, L. japonica and L. similes seem to be similar, resulting in the difficulty of clarifying the origins of Flos Lonicerae solely by HPLC profiles of saponins, in addition to the clear difference of the HPLC profiles of saponins from L. macranthoides and L. hypoglauca.Therefore, in addition to the conventional morphological and histological identification methods, the contents and the HPLC profiles of saponins and iridoids could also be used as accessory chemical evidence toclarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.4. ConclusionsThis is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside in Flos Lonicerae. Together with the HPLC profiles of iridoids, the HPLC-ELSD profiles of saponins could also be used as an accessory chemical evidence to clarify the botanical origin and comprehensive quality evaluation of Flos Lonicerae.AcknowledgementsThis project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program（2003AA2Z2010）.[1]Ministry of Public Health of the People’s Republic of China, Pharmacopoeia ofthe People’s Republic of China, V ol.1, 2000, p. 177.[2]W. Shi, R.B. Shi, Y.R. Lu, Chin. Pharm. J., 34(1999) 724.[3]J.B. Xing, P. Li, D.L. Wen, Chin. Med. Mater., 26(2001) 457.[4]Y.Q. Zhang, L.C. Xu, L.P. Wang, J. Chin. Med. Mater., 21(1996) 204.[5] D. Zhang, Z.W. Li, Y. Jiang, J. Pharm. Anal., 16(1996) 83.[6]T.Z. Wang, Y.M. Li, Huaxiyaoxue Zazhi, 15(2000) 292.[7]J.ZH. Shi, G.T. Liu. Acta Pharm. Sin., 30(1995) 311.[8]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 209.[9]Y. P. Liu, J. Liu, X.SH. Jia, et al. Acta Pharmacol. Sin., 13 (1992) 213.[10]J.ZH. Shi, L. Wan, X.F. Chen.ZhongYao YaoLi Yu LinChuang, 6 (1990) 33.[11]J. Liu, L. Xia, X.F. Chen. Acta Pharmacol. Sin., 9 (1988) 395[12]H.J. Li, P. Li, W.C. Ye, J. Chromatogr. A 1008(2003) 167-72.[13]Q. Mao, D. Cao, X.SH. Jia. Acta Pharm. Sin., 28(1993) 273.[14]H. Kizu, S. Hirabayashi, M. Suzuki, et al. Chem. Pharm. Bull., 33(1985) 3473.[15]S. Saito, S. Sumita, N. Tamura, et al. Chem Pharm Bull., 38(1990) 411.[16]Alltech ELSD 2000 Operating Manual, Alltech, 2001, p. 16. In Chinese.[17]J.B. Xing, P. Li, Chin. Med. Mater., 22(1999) 366.Fig. 1 Chemical structures of seven saponins from Lonicera confusa macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-D-glucopyranosyl(6→1)-O-β-D- glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-L-arabinopyranosyl(2→1)-O-α-L-rhamnopyranoside (7)Fig. 2Representative HPLC chromatograms of mixed standards and methanol extracts of Flos Lonicerae.Column: Agilent Zorbax SB-C18 column（250 4.6mm, 5.0µm）, temperature of 25℃; Detector: ELSD, drift tube temperature 106℃, nitrogen flow-rate 2.6 l/min.A: Mixed standards, B: L. confusa, C: L. japonica, D: L. macranthoides, E: L. hypoglauca, F: L. similes.Table 1 Calibration curves for seven saponinsAnalytes Calibration curve ar2Test range(μg)LOD(μg)LOQ(μg)1 y=6711.9x－377.6 0.9940 0.56–22.01 0.26 0.882 y=7812.6x－411.9 0.9922 0.54–21.63 0.26 0.843 y=6798.5x－299.0 0.9958 0.46–18.42 0.22 0.724 y=12805x－487.9 0.9961 0.38–15.66 0.10 0.345 y=4143.8x－88.62 0.9989 0.42–16.82 0.18 0.246 y=3946.8x－94.4 0.9977 0.40–16.02 0.16 0.207 y=4287.8x－95.2 0.9982 0.42–16.46 0.12 0.22a y: Peak area; x: concentration (mg/ml)Table 2 Reproducibility of the assayAnalyteIntra-day variability Inter-day variability Content (mg/g) Mean RSD (%) Content (mg/g) Mean RSD (%)1 46.1646.2846.2246.22 0.1346.2245.3647.4226.33 2.232 5.385.385.165.31 2.405.285.345.045.22 3.043 4.374.304.184.28 2.244.284.464.024.255.204 nd1)-- -- nd -- --5 1.761.801.821.79 1.701.801.681.841.77 4.706 1.281.241.221.252.451.241.341.201.26 5.727 tr2)-- -- tr -- -- 1): not detected; 2): trace. RSD (%) = (SD/Mean) ×100%Table 3 Recovery of the seven analytesAnalyteOriginal(mg) Spiked(mg)Found(mg)Recovery(%)Mean(%)RSD(%)1 23.0823.1423.1119.7122.8628.1042.7346.1351.0199.7100.699.399.8 0.722.692.672.582.082.913.164.735.515.7698.197.6100.698.8 1.632.172.152.091.732.182.623.884.404.6598.8103.297.799.9 2.94nd1)1.011.050.980.981.101.0297.0104.8104.1102.0 4.250.880.900.910.700.871.081.561.752.0197.197.7101.898.9 2.660.640.620.610.450.610.751.081.211.3397.796.796.096.8 0.97tr2)1.021.101.081.031.111.07100.9102.799.1100.9 1.81): not detected; 2): trace.a Recovery (%) = (Amount found –Original amount)/ Amount spiked ×100%, RSD (%) = (SD/Mean) ×100%Table 4 Contents of seven saponins in Lonicera spp.Content (mg/g)1 2 3 4 5 6 7 L. confusa45.65±0.32 5.13±0.08 4.45±0.11tr1) 2.04±0.04tr 1.81±0.03 L. japonica nd2)nd 3.44±0.09nd nd nd nd L. macranthoides46.22±0.06 5.31±0.13 4.28±0.10 tr 1.79±0.03 1.25±0.03 tr L. hypoglauca11.17±0.07 nq3)53.78±1.18nd 1.72±0.02 2.23±0.06 2.52±0.04 L. similes41.22±0.25 4.57±0.07 3.79±0.09nd 1.75±0.02tr nd 1): trace; 2): not detected.. 3) not quantified owing to the suspicious purity of the peak.。

Quality of medicines questions and answers These questions and answers address a number of questions that have been brought to the attention of the Joint Committee for Medicinal Products for Human Use / Committee for Medicinal Products for Veterinary Use Quality Working Party (QWP) by marketing-authorisation holders (MAHs) or European Economic Area (EEA) competent authorities, on matters related to the quality of medicines. They have been developed and are maintained by the QWP.They provide the EEA's harmonised position on issues that can be subject to different interpretation or require clarification, typically arising from discussions or correspondence during assessment procedures.If a question is not addressed, marketing-authorisation holders are encouraged to contact the European Medicines Agency for further information atqwp@ema.europa.eu.These questions have been produced to provide clarification or additional information, and should be read in conjunction with the European Pharmacopoeia, quality guidelines and other guidance documents.Key:∙H: applicable to medicinal products for human use∙V: applicable to veterinary medicinal productsQuality of medicines questions and answers: Part 1：Active Substance - Active-substance-master-file procedure1.Can a mixture of an active substance with an excipient be submitted through anactive-substance-master-file (ASMF) procedure? H+V August 2007.No. A mixture of an active substance with an excipient cannot be submitted through an ASMF procedure.The blending of an active substance and an excipient is considered as the first step in the manufacture of the medicinal product, and therefore does not fall under the definition of an active substance.The only exceptions can be made where the active substance cannot exist on its own,for example, due to insufficient stability without a stabilising agent, or in the case of herbal dry extracts if it is not possible to produce a solid extract without excipients. Active substance - Declaration by the qualified person on the good-manufacturing-practice status of the active substance manufacturer1.The notice to applicants requires the submission of a declaration signed by thequalified person that the active substance used is manufactured in accordance with good manufacturing practice. The active substance in my product is widely used, but not normally as a pharmaceutical active substance, and I am having some difficulty in confirming compliance. What should I do to furnish the required declaration? H+V September 2008Full compliance with good manufacturing practice (GMP) for finished products and active substances is a legal obligation for manufacturing-authorisation holders. It is recognised that for a small number of medicinal products the primary use of the active substance is not in a medicinal product and the producer may therefore not be aiming to meet the specific requirements of pharmaceutical customers that represent an insignificant volume of business. Alternative sources should normally be sought but in exceptional circumstances the manufacturing-authorisation holder should assess and document to which extent GMP is complied with and provide a risk-based justification for the acceptance of any derogation. The declaration provided by the qualified person should set out in detail the basis for declaring that the standards applied provide the same level of assurance as GMP. The European Medicines Agency will collect experience with this approach which can be used as a basis for discussion on related amendments to guidelines in the future.Active Substance - Good-manufacturing-practice compliance for sterilisation of an active substance1.What kind of good-manufacturing-practice documentation is needed for an activesubstance manufacturer who performs sterilisation of an active substance? H+V July 2010The good-manufacturing-practice (GMP) basic requirements for active substances used as starting materials (European Union (EU) GMP guide part II) only applies to the manufacture of sterile active substances up to the point immediately prior to the active substance being rendered sterile. The sterilisation and aseptic processing of sterile active substances are not covered by this guideline and shall be performed in accordance with GMP for medicinal products (Commission Directive 2003/94/EC; as interpreted in the basic requirements for medicinal products including annex 1 of theEU GMP Guide part I). This implies that for any active-substance manufacturer who performs sterilisation and subsequent aseptic handling of the active substance, a valid manufacturing authorisation or GMP certificate from an EEA authority or from an authority of countries where mutual recognition or other Community arrangements apply has to be submitted.Also, the active-substance manufacturer has to submit data on the sterilisation process of the active substance (including validation data) to the marketing-authorisation applicant or holder for inclusion in the dossier submitted for the finished product and approval by the licensing authority or authorities.Active Substance - Starting materials of herbal origin1.How should the quality of a starting material of herbal origin be controlled when itis used to manufacture a semi-synthetic active substance? H+V February 2012 There are several types of starting materials of herbal origin used to manufacture semi-synthetic active substances:herbal drugs;herbal preparations (typically extracts);isolated plant constituents (no additional synthetic steps).Starting materials of herbal origin should be characterised to ascertain their suitability and a contaminant profile should be established and submitted, taking into consideration the number of chemical steps between the starting material and the semi-synthetic active substance.In all cases, the scientific name (genus, species, variety and author) of the plant and plant part used should be specified. If the starting material is an extract or an isolated constituent, the extraction solvent and strength (e.g. ethanol 50% v/v) used for the first extraction from the herbal drug should be specified as well.The quality of the starting material of herbal origin should follow the principles set out in the European Pharmacopoeia monographs on herbal drugs, herbal drug preparations, extracts and essential oils, as applicable: the potential presence of foreign matter, pesticides, microbiological contamination, total ash, heavy metals, aflatoxins, ochratoxin A, radioactive contamination, residual solvents, and other relevant impurities should be discussed.Potential contaminants that may be carried through the extraction and purification processes should be fully addressed taking into account the production of the herbal drug, and the subsequent extraction and purification processes.The specification for the starting material of herbal origin should be fully justified by the applicant and should include suitable tests for identity, assay, impurities and potential contaminants.Compliance with the guideline on good agricultural and collection practice (GACP) is not mandatory in the steps prior to the starting material of a semi-synthetic active substance, since it applies to herbal medicinal products and traditional herbal medicinal products. However, information on the geographical origin, collection or cultivation, harvesting, and post-harvest treatments (possible pesticides and fumigants used and possible radioactive contamination) could justify limited testing for (possible) contaminants.Data submission1.Is the submission of data regarding manufacturing process validation, analyticalvalidation, and stability studies, produced only by the developer of the product sufficient? Does it make any difference if the developer of the product is presented as one of the product manufacturers or not? H+V July 2008These questions and answers only concern products that are oral solid dosage forms manufactured by standard manufacturing methods. The new product has the same formula and manufacturing method of the developed product.Provided that the formula, manufacturing process, analytical methods and packaging materials are the same, data originating from the developer of the product is normally sufficient. As regards manufacturing process validation, the marketing authorisation holder, according to the guideline on process validation, must submit with the new application the process validation scheme and the commitment to carry out process validation and initiate stability studies along with the batch analysis for production scale batches for the new manufacturing site. This is irrespective of whether the product developer is one of the manufacturing sites of the new product or not.2.Who is responsible to verify the production scale validation data when this isavailable? H+V July 2008These questions and answers only concern products that are oral solid dosage forms manufactured by standard manufacturing methods. The new product has the same formula and manufacturing method of the developed product.According to the guideline on process validation, "the results can be subsequently verified by supervising authority according to national procedure.” Depending on the product and the concerns that may arise, in some countries this may be dealt with as a post-authorisation commitment or it may be brought to the attention of good-manufacturing-practice inspectors to be checked during their next inspection. The marketing-authorisation holder is usually not expected to present these data with the new application, unless it is requested by the licensing authorities.3. Can the test product of a bioequivalence study be produced by the developer, irrespective of whether that developer is one of the manufacturing sites of the new product? H+V July 2008These questions and answers only concern products that are oral solid dosage forms manufactured by standard manufacturing methods. The newproduct has the same formula and manufacturing method of the developed product. The product used in the bioequivalence study must be representative of the industrial scale product to be marketed.A bioequivalence study conducted using a test product produced by the developer is acceptable if the developer is also one of the manufacturers of the new product.If the developer is not one of the manufacturers of the new product, it has to be demonstrated that the bioequivalence batch is representative of the industrial scale product to be marketed.For this the following applies:The developer product and the new product must have identical formula, specifications, manufacturing process and equipment;The active substance supplier is the same or it is ensured that characteristics of the active substance that may have an impact on the bioavailability are identical;The excipients characteristics which may have an impact on the bioavailability of the active substance are identical;The dissolution profiles of batches produced by the developer (including the biobatch) and the product from the new manufacturer (at least pilot batches) must be similar;Comparative pilot batch analysis between the developer product (including the biobatch) and the new manufacturer product must be presented.4. Is there any requirement for the marketing-authorisation holder to submit any kind of proof (e.g. statement or contract) that it has obtained the dossier from the developer legally? H+V July 2008These questions and answers only concern products that are oral solid dosage forms manufactured by standard manufacturing methods. The new product has the same formula and manufacturing method of the developed product. No such proof is necessary to be presented in the application dossier.European Pharmacopeia (Ph. Eur.) - Harmonised Ph. Eur. Chapters 2.6.12, 2.6.13 and 5.1.41.How should any update to the microbial quality control of a finished product,specifically linked to the update to the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) harmonised Ph. Eur. methods (2.6.12 and 2.6.13) and limits (5.1.4) be achieved? H+V February 2012Any changes to implement the harmonised methods (2.6.12 and 2.6.13) should be submitted under change code B.II.d.2.a (type IA).Any changes to implement the harmonised limits (5.1.4) should be submitted asfollows:Changes to the limits – B.II.d.1.a (type IA), but only in the following situations:The currently registered microbial control limits (present situation) are totally in line with the pre-January 2008 (non-harmonised) situation and did not include any additional specified controls over and above the Pharmacopoeia requirements for that particular dosage form. In addition, the proposed controls are totally in line with the harmonised monograph.If for any reason these criteria are not met, a type IB variation (B.II.d.1.z) should be submitted.In accordance with current requirements, any changes should be clearly identified (present / proposed) in the variation submission and the affected parts of the marketing-authorisation dossier should be appropriately updated, including a description of how the method is applied to the specific product.Please note that this advice supersedes the questions and answers dated August 2007, which are now out of date.European Pharmacopeia - Monograph on tablets1.How should industry apply the revised test subdivision of tablets in the EuropeanPharmacopeia monograph on tablets? H+V October 2006The requirements of the European Pharmacopoeia (Ph. Eur.) general monograph on tablets for tablets with one or more breaklines have been revised as of 1 July 2006. The previous version of the monograph, which has been in force since April 2002, requested compliance with either test A (uniformity of content of single-dose preparations) or the test for uniformity of mass of single-dose preparations. It did not, however, give any information on how to select the parts to be tested. Supplement 5.5 now provides background information on the use of breakmarks and describes the sampling procedure and the number of tablet parts to be tested in detail. As a test procedure, it prescribes determining uniformity of mass, while setting the somewhat wider acceptance criteria of the test of content uniformity, namely 85 - 115% (in this case of the average mass).According to Directives 2001/83/EC and 2001/82/EC, the monographs of the Ph. Eur. are the official standards of appropriate quality in the marketing authorisation procedures. In the pharmacopoeia it is also stated that a preparation must comply with the monograph throughout its period of validity.As the revised text defines lesser requirements with wider acceptance criteria than the previous version (see above), a decision has been taken by the regulatory authorities of the European Union (EU) that the revised test on subdivision of tablets is to be applied to all new applications for marketing authorisations as well as all existingproducts. It is acknowledged, however, that the new test will not have been applied to products which are in the final stages of their development. In order not to delay any immediate applications and in line with the period of time defined in the variations regulation, a transitional period of 6 months following the coming into force of Ph. Eur. 5.5 has been defined, in which the EU regulatory authorities will accept submission of results which have been generated using the previous version of the monograph.As the requirements on subdivision of tablets are listed in the production section of the general monograph on tablets, it will normally be sufficient to perform the test only in the framework of pharmaceutical development. There is no need to include the test in the release specification. However, in situations where there is a significant change in tablet hardness during storage, it may be necessary to repeat the test at the end of shelf-life in order to ensure that the scorability has not changed as a function of hardness.European Pharmacopeia (Ph. Eur.) - Harmonised chapter uniformity of dosage unitsBackground. H+V June 2012Between 2004 and 2008, various questions and answers have been published on this website to clarify how the harmonised European Pharmacopoeia (Ph. Eur.) chapter on uniformity of dosage units (2.9.40) and the general chapters on uniformity of mass of single-dose preparations (2.9.5) and uniformity of content of single-dose preparations (2.9.6) should be applied in the European Union (EU), also in view of the compliance objection made by the United States Food and Drug Administration to the USP on the 2% relative-standard-deviation clause in general chapter 2.9.40. This matter has been recently reviewed by EU regulators and the resulting agreed position is presented in the following questions and answers, which replace the previous questions and answers 1 and 4 published on the same subject in 2004 and 2006, which have now been deleted from the website.1.How should industry demonstrate compliance with the European Pharmacopoeiawith regard to uniformity of dosage units? H+V June 2012Medicinal products marketed in the European Union need to be in compliance with the relevant requirements in the European Pharmacopoeia (Ph. Eur.).From a pharmaceutical quality point of view, the approach taken in the harmonised general chapter on uniformity of dosage units (2.9.40) is considered equivalent to what was previously required in the Ph. Eur. through the general chapters on uniformity of mass of single-dose preparations (2.9.5) and uniformity of content of single-dose preparations (2.9.6). These general chapters, 2.9.5 and 2.9.6, are still included in the current version of the Ph. Eur.Taking this into account, the decision on what approach to take is left to the applicant. Application of either the Ph. Eur. harmonised general chapter on uniformity of dosage units (2.9.40) or the Ph. Eur. general chapters on uniformity of mass of single-dose preparations (2.9.5) and uniformity of content of single-dose preparations (2.9.6) are both considered acceptable options to demonstrate compliance with the Ph. Eur. with regard to uniformity of dosage units.Please note that this advice supersedes the previous questions and answers 1 and 4 published on the same subject in 2004 and 2006. The remaining previously published questions and answers on the use of the general chapter 2.9.40 (former questions and answers 2, 3, 5 and 6) are still valid and remain published as questions and answers 2 to 5.2. How should the new text be interpreted, in particular in relation to testing frequency (routine test / skip test / test on validation batches only)? H+V March 2006 Where a dosage form monograph in the European Pharmacopoeia refers to this general chapter, the product should comply. This is normally verified by routine testing unless otherwise justified and authorised.3. How can the text be interpreted in light of the compliance objection made by the Food and Drug Administration to the United States Pharmacopeia on the 2% relative standard deviation clause? H+V March 2006The harmonised text is included in the European Pharmacopoeia and the 2% clause will thus be valid in the European Union. This will not be dependent on the final outcome of the discussion between the Food and Drug Administration and the United States Pharmacopeia.4. When can the requirements in the harmonised European Pharmacopoiea general chapter on uniformity of dosage units (2.9.40) for mass variation, rather than content uniformity, be applied with reference to a relative standard deviation (RSD) of not more than 2%? Is it sufficient to determine the RSD on for example 3 validation batches? H+V July 2008It is the opinion of the Quality Working Party that the requirement in the harmonised chapter 2.9.40 regarding the 2% relative-standard-deviation clause represents minimum requirements. The use of this clause should be based on sufficient experience. Normally, results from 3 validation batches are not sufficient. It is rather to be used post-approval when more extensive production experience is gained.5. What is meant by single component and multiple component in the harmonised chapter 2.9.40? H+V July 2008'Single component' means one active substance with no excipient. 'Multiple component' could mean 'at least 2 active substances' or 'at least 1 active substance and at least one excipient'; both cases are valid.Impurities - Calculation of thresholds for impurities1.What is the basis for the calculation of thresholds to set limits for impurities in thefinished product specification? H July 2009The calculated thresholds should be based on the highest maximum daily dose of the respective active substance in authorised medicinal products. The threshold for impurities should be the same for all strengths.The same rule applies for combination drugs; the highest possible combined strength should be used for setting the thresholds.The maximum daily dose used for threshold calculation should be the same for a given active substance irrespective of the medicinal product.Impurities - Harmonisation of policies on setting specifications for potentially genotoxic impurities, heavy-metal-catalyst residues and class-1 solvent residues1.What is a reasonable policy for setting specifications for potentially genotoxicimpurities which are theoretical or actual impurities in a drug substance manufacturing process? H June 2012Different possible scenarios can be identified and the applicable policies to be applied for each of them are described below:Example 1 – A potential genotoxic impurityThe definition for a potential genotoxic impurity is derived from the definition for 'potential impurity': an impurity that theoretically can arise during manufacture or storage. It may or may not actually appear in the (new) drug substance (ICH Q3A, glossary).If a potential genotoxic impurity is just a theoretical impurity i.e. based on theoretical considerations but not found in practice at any key stage in the manufacturing process as demonstrated by studies during development of the manufacture, the impurity does not need to be included in the drug substance specification or a specification of an intermediate.Example 2 – A (potentially) genotoxic impurity actually formed or introduced prior to the final step of the synthesisIf a (potentially) genotoxic impurity is formed or introduced in a step before the final synthesis step, it is considered possible to not include this impurity in the drug substance specification if it is controlled by a suitable limit in a synthesis intermediate and if it is unambiguously demonstrated by analysis results (use of spiking experiments are encouraged) that presence of this impurity does not exceed 30 % of the limit, derived either from threshold of toxicological concern (TTC) or otherwisedefined acceptable limit etc., in the drug substance.It is considered possible to apply skip testing if the level of the impurity in a synthesis intermediate does not exceed 30% of the limit, derived from either TTC or otherwise defined acceptable limit etc, in the intermediate. Data should be presented for at least 6 consecutive pilot scale or 3 consecutive production scale batches. If this condition is not fulfilled, a routine test in the intermediate is needed. If the impurity exceeds 30% of the limit, derived either from TTC or otherwise defined acceptable limit etc., in the drug substance the impurity has to be included in the drug substance specification and the test has to be carried out on a routine basis.Should a genotoxic impurity not be controlled at the intermediate stage, then the scenario of example 3 applies.Example 3 - A (potentially) genotoxic impurity is formed or introduced in the last synthesis stepIf a (potentially) genotoxic impurity is formed or introduced in the final synthesis step, it should be included in the specifications. However, it is considered possible to apply skip testing if the level of the impurity does not exceed 30% of the limit, derived from either TTC or otherwise defined acceptable limit etc., in the drug substance. Data should be presented for at least 6 consecutive pilot scale or 3 consecutive production scale batches. If this condition is not fulfilled, a routine test in the drug substance specification is needed.Definitions:For the purpose of these questions and answers, the following definitions apply: Genotoxic impurity: an impurity that has been demonstrated to be genotoxic in an appropriate genotoxicity test model, e.g. bacterial gene mutation (Ames) test.Potentially genotoxic impurity: an impurity that shows (a) structural alert(s) for genotoxicity but that has not been tested in an experimental test model. Here potentially relates to genotoxicity, not to the presence or absence of this impurity.2.In order to harmonise the policies to be applied for setting specifications forgenotoxic impurities and heavy-metal-catalyst residues, what are reasonable policies to be applied when setting specifications for heavy-metal-catalyst residues? H June 2012In order to harmonise the policy for setting specifications for metal residues with that for setting specifications for genotoxic impurities, some clarifications of the interpretation of sections 4.5 and 4.6 of the heavy-metal-catalyst guideline (EMEA/CHMP/SWP/4446/2000) are given below.Since it is the class-1 metals that are the most toxic metals with permitted dailyexposures (PDEs) that approach the level of the threshold of toxicological concern (TTC) applied for genotoxic impurities, it seems reasonable that class-1 metals are the prime focus for harmonisation with the policy for genotoxic impurities while class-2 and 3 metals could be treated similarly but somewhat less strictly.Example 1 –A class-1 metal is not used or suspected to be present in a synthesis processMetals are not expected to be formed in synthesis processes. Only if deliberately introduced or suspected to be present for other reasons, residues of metals can be expected. If not used or suspected to be present, the metal does not need to be included in the drug substance specification or a specification of an intermediate.Example 2 –A class-1 metal is formed or introduced prior to the final step of the synthesisIf a class-1 metal is introduced in a step before the final synthesis step, it is considered possible to not include this metal in the drug substance specification if it is controlled by a suitable limit in a synthesis intermediate and if it is unambiguously demonstrated by analysis results that the presence of this metal does not exceed 30% of the guideline limit in the drug substance.It is considered possible to apply skip testing if the level of the class-1 metal in a synthesis intermediate does not exceed 30% of the guideline limit in the intermediate. Data should be presented for at least 6 consecutive pilot scale or 3 consecutive production scale batches. If this condition is not fulfilled, a routine test in the intermediate is needed. If the class-1 metal exceeds 30% of the guideline limit in the drug substance the impurity has to be included in the drug substance specification and the test has to be carried out on a routine basis.Should a class-1 metal not be controlled at the intermediate stage, then the scenario of example 3 applies.Example 3 – A class-1 metal is introduced in the last synthesis stepIf a class-1 metal is introduced in the final synthesis step, it should be included in the specifications. However, it is considered possible to apply skip testing if the level of the metal does not exceed 30% of the guideline limit in the drug substance. Data should be presented for at least 6 consecutive pilot scale or 3 consecutive production scale batches. If this condition is not fulfilled, a routine test in the drug substance specification is needed.3.In order to harmonise the policies to be applied for setting specifications forgenotoxic impurities and class-1 solvent residues, what are reasonable policies to be applied when setting specifications for class-1 solvent residues? H June 2012。

Obtain……an adequate sampling from the ectocervix using a plastic spatula.Rinse……the spatula as quickly as possible into the PreservCyt®Solutionvial by swirling the spatula vigorously in the vial 10 times.Discard the spatula.Obtain……an adequate sampling from the endocervix using an endocervicalbrush device. Insert the brush into the cervix until only the bottom-most fibers are exposed. Slowly rotate 1/4 or 1/2turn in onedirection. DO NOT OVER-ROTATE.Rinse……the brush as quickly as possible in the PreservCyt Solution byrotating the device in the solution 10 times while pushing againstthe PreservCyt vial wall. Swirl the brush vigorously to furtherrelease material. Discard the brush.Tighten……the cap so that the torque line on the cap passes the torque lineon the vial.Record……the patient’s name and ID number on the vial.…the patient information and medical history on the cytologyrequisition form.Place……the vial and requisition in a specimen bag for transport tothe laboratory.Endocervical Brush/Spatula ProtocolPart No.85217-002 Rev. HThe Test You TrustObtain……an adequate sampling from the cervix using a broom-like device. Insert the central bristles of the broom into the endo-cervical canal deep enough to allow the shorter bristles to fully contact the ectocervix. Push gently , and rotate the broom in a clockwise direction five times.Rinse……the broom as quickly as possible into the PreservCyt ®Solution vial by pushing the broom into the bottom of the vial 10 times, forcing the bristles apart. As a final step, swirl the broom vigorously to further release material. Discard the collection device.Tighten……the cap so that the torque line on the cap passes the torque line on the vial.Record……the patient’s name and ID number on the vial.…the patient information and medical history on the cytology requisition form.Place……the vial and requisition in a specimen bag for transport to the laboratory .Broom-Like Device Protocol。

WHO Model Formulary for ChildrenBased on the Second Model List of Essential Medicines for Children 2009世界卫生组织儿童标准处方集基于2009年儿童基本用药的第二个标准目录WHO Library Cataloguing-in-Publication Data:WHO model formulary for children 2010.Based on the second model list of essential medicines for children 2009.1.Essential drugs.2.Formularies.3.Pharmaceutical preparations.4.Child.5.Drug utilization. I.World Health Organization.ISBN 978 92 4 159932 0 (NLM classification: QV 55)世界卫生组织实验室出版数据目录：世界卫生组织儿童标准处方集基于2009年儿童基本用药的第二个标准处方集1．基本药物 2.处方一览表 3.药品制备 4儿童 5.药物ISBN 978 92 4 159932 0 (美国国立医学图书馆分类：QV55)World Health Organization 2010All rights reserved. Publications of the World Health Organization can be obtained fromWHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: ******************). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the aboveaddress(fax:+41227914806;e-mail:*******************).世界卫生组织2010版权所有。