作者感谢 Schütze 博士为他的帮助建立了提出的方法和博士 Zoller 为有价值的输入有关正确的校准范围。作者还认识到质谱设备的技术人员。

注: 建议在处理有机溶剂时, 如甲醇, 在通风罩工作。准备所有的缓冲和移动阶段的容积烧瓶。如未另行规定, 溶液可在室温下贮存1月。
1. 校准仪和质量控制样品的编制
注: 在补充文件中给出了用于准备库存和穗状溶液的相应数据分析表。出于可追溯性的原因, 请在相应的列中插入制造商、目录编号和每种抗生素的大量数量。将所有抗生素溶解在4摄氏度的冷藏库中, 尽量缩短工作时间。
<ol><li>在水中准备100毫升25% 甲醇: 预先填充100毫升容积烧瓶, 含25毫升的绝对甲醇, 用蒸馏水将其填满100毫升。</li>
	<li>在水中准备10毫升200毫米醋酸: 预先填充10毫升容积烧瓶, 含9毫升的 HPLC 级水, 加入115µL 的冰乙酸 (99.5% 纯度, 17.4 米), 并加蒸馏水高达10毫升。</li>
	<li>在水中准备25毫升的25% 甲醇, 用20毫米醋酸: 预先填充一个25毫升容积烧瓶, 含2.5 毫升的水200毫米醋酸溶液, 加入6.25 毫升的绝对甲醇, 用蒸馏水填满瓶子到25毫升。</li>
	<li>使用精确刻度来衡量15毫升锥形管中适当数量的抗生素, 如列初始重量中补充文件所述。</li>
	<li>在25% 甲醇水中制备氟喹诺酮类、环丙沙星和莫西沙星的库存溶液, 包括20毫米醋酸。为此, 请将相应的卷添加到加权数量中, 如 "最终卷" 列中的补充文件所述。在超声浴中迅速溶解喹诺酮抗生素2分钟, 并通过强烈的涡流。</li>
	<li>在25% 甲醇水中制备头孢吡肟、美罗培南、利奈和哌拉西林的库存溶液。为此, 将相应的卷添加到列最终卷中的补充文件中所述的加权数量, 并通过强烈的涡流快速溶解抗生素。将美罗培南溶解为最后一种物质。</li>
	<li>将所述补充文件中相应的库存溶液图中所描述的所有抗生素的库存解决方案组合在一起, 以产生多峰集中的穗型溶液。</li>
	<li>穗九卷无药血清与一卷的0–7浓缩穗解决方案, 以获得血清校准和质量控制 (QC) A–D。例如, 在10毫升的聚丙烯管中加入0.5 毫升的穗溶液, 在4.5 毫升的血清中, 在 4 rpm 的滚筒搅拌机上, 在50摄氏度的冷库中孵化15分钟。</li>
	<li>使用重复的吸管产生100µL 整除数的校准和 QCs 在1.5 毫升聚丙烯管。</li>
	<li>将校准、质量控制和抗生素库存解决方案存储在摄氏-80 摄氏度, 长达六月。</li>
	<li>对于每种抗生素, 也准备一个整洁的解决方案, 含有1000毫克/升的单一抗生素。用适当的稀释剂稀释相应的库存溶液 (例如, 对环丙沙星, 使用25% 甲醇水, 包括20毫米醋酸)。 注意: 只有在仪器调谐时才需要整洁的抗生素解决方案。</li>
</ol>2. 内部标准组合的编制
注: 内部标准是在样品清理过程中添加到样品中的感兴趣分析物的同位素标记对应物。由于内部标准与未标记的对应物具有几乎相同的整体物理化学性质, 它们补偿了给定样本的矩阵效应。
<ol><li>10毫升50% 甲醇在水中添加5毫升的绝对甲醇到10毫升摇瓶, 并填补它高达10毫升与蒸馏水。</li>
	<li>在水中制备10毫升50% 甲醇, 包括20毫米醋酸。要做到这一点, 添加1毫升的200毫米醋酸到10毫升瓶, 添加5毫升的绝对甲醇, 并填补它高达10毫升与蒸馏水。</li>
	<li>用1000毫克/升直接在制造商提供的瓶子中生成内部标准的库存解决方案。溶解头孢吡肟-13C12d-3硫酸盐在蒸馏水, 美罗培南 d6, 利奈 d3, 和哌拉西林 d5在50% 甲醇水解答。将环丙沙星8在50% 甲醇水中溶解, 20 毫米醋酸盐和盐酸莫西沙星-13C1D3在蒸馏水与20毫米醋酸盐。</li>
	<li>结合的是在1.5 毫升聚丙烯管的库存解决方案, 以产生一个高浓度的内部标准混合。添加10µL 头孢吡肟-13C12D3, 10 µL 的美罗培南 d 6, 1 µL 的环丙沙星 d8, 2 µL 盐酸莫西沙星-13C1D3, 2 µL 利奈 D3, 10 µL 哌拉西林-D5到965µL 25% 甲醇水。</li>
	<li>存储内部标准库存解决方案, 并将其浓缩为-80 摄氏度。</li>
</ol>3. 病人样本储存
注: 确保血清获得尽可能快, 冷冻样品的冷链保持。
<ol><li>收集血清收集管中的全血。</li>
	<li>在室温下让血凝块20–30分钟。</li>
	<li>将血清从血液中分离出来, 离心 2000 x克, 10 分钟。</li>
	<li>将上清液转移到干净的聚丙烯管上。</li>
	<li>将血清贮存六月至-80 摄氏度, 直至化验。或者, 将样品储存在-20 摄氏度3天。</li>
</ol>4. 色谱缓冲剂的制备
<ol><li>在水中制备1米甲酸铵, 用100毫升的摇瓶将甲酸铵的6.306 克溶解于100毫升的 HPLC 级水中。将解决方案存储在4摄氏度的1月。</li>
	<li>准备移动相 A [10 毫米甲酸铵在水甲酸 (99.9: 0.1 伏/五)]。预先填充1000毫升容积烧瓶, 约500毫升的 HPLC 级水, 添加1毫升甲酸和10毫升的1米甲酸铵溶液, 并填充到1000毫升的 hplc 级水。将移动相 a 转移到干净的玻璃瓶上, 并将其连接到高效液相色谱系统。在室温下存储移动相2周。</li>
	<li>准备移动阶段 b. 将 HPLC 级绝对甲醇转化为清洁玻璃瓶, 并将其与 hplc 系统连接。</li>
	<li>使用绝对甲醇作为洗针溶剂, 并将相应的管连接到含有移动相 B 的玻璃瓶中。</li>
	<li>生成甲醇-水甲酸 (7:92.9: 0.1, v/v/v) 的密封和净化溶剂。预先填充1000毫升容积烧瓶约500毫升蒸馏水, 添加70毫升的绝对甲醇, 1 毫升甲酸, 并添加蒸馏水到1000毫升。将溶剂转移到干净的玻璃瓶上, 并将其与高效液相色谱系统连接。 注: 各种 autosampler 系统使用强和弱针洗溶剂。在这种情况下, 根据制造商的建议准备洗涤解决方案。例如, 用甲醇-水-isopropylic 酒精 (70:20:10, v/v/v) 和弱洗涤与水-甲醇 (95:5, v/v) 进行强洗涤。</li>
</ol>5. 仪器调谐
注意: 此步骤是为在特定质谱仪上的方法的设置而执行的。
<ol><li>稀释1000毫克/升分析物和内部标准溶液1:10 或1:100 在混合的移动相 a 和 B (50:50, v/v), 取决于探测器信号强度。用 autotune 函数调整质谱仪或对以下父-子离子转换进行手动调整14: 头孢吡肟 (481.0 > 167.0/395.7), 头孢肟-13C12D3 (485.1 > 167.1/400.0), 美罗培南 (384.1 > 114.0/141.0), 美罗培南 D6 (390.1 > 114.0/147.2), 环丙沙星 (332.0 > 231.0/245.0), 环丙沙星 D8 (340.1 > 235.1/249.3), 莫西沙星 (402.0 > 261.0/383.9), 莫西沙星-13C1D3 (406.1 > 265.1/388.0), 利奈 (338.0 > 235.0/296.0), 利奈 D3 (341.1 > 235.1/297.1), 哌拉西林 (518.0 > 143.0/358.9), 和哌拉西林 D5 (523.1 > 142.8/364.1)。</li>
	<li>对于具有自动调谐的仪器, 使用 autotune 函数通过探测器自动调整 MS 进气口的电压和设置。</li>
	<li>对于具有手动调谐的仪器, 请调整设置 (例如, 碰撞电压和碰撞能量), 直到在探测器上为每个母和子离子获得最佳 (通常为最大) 信号强度。例如, 插入混合三通, 将移动相 a 和 B (50:50, v/v) 放在0.5 毫升/分钟, 并持续注入整齐的抗生素或内部标准, 流速为0.1 毫升/分钟。</li>
</ol>6. 高效液相色谱-ms/ms 设置
注: 质谱仪、HPLC 系统 (包括 autosampler) 的特点, 以及相应的软件依赖于制造商。根据制造商的建议, 调整质量光谱仪参数和洗涤程序。
<ol><li>将质谱仪参数存储在相应的 "MS 调谐文件"中。使用电喷雾电离在正模式 (ESI +) 为所有的方法。调整所用仪器的离子源设置 (例如, 毛细管电压为1.5 伏, 源温度为120摄氏度, desolvation 温度为400摄氏度, desolvation 气体流速为600升/小时, 射频透镜电压为 0.1 V, 停留时间为80毫秒)。</li>
	<li>在 "MS 文件"中指定分析物和内部标准调谐参数 (如毛细管电压、碰撞能量)。</li>
	<li>在 "入口文件"中设置以下 autosampler 条件: 样品温度在10°c 以5°c 的极限;洗涤顺序在1x 清洗-洗涤-清洗与600µL 清除容量替换。</li>
	<li>在上述 '入口文件 ', 设置流量为0.4 或0.5 毫升/分钟, 运行时间为4分钟, 压力高限制到345条, 和柱温度到30°c 与极限的 @ 5 °c。添加流动相 A 和 B 的溶剂名称, 分别设置为 7% B/93%a。</li>
	<li>程序在 '入口文件 '的色谱梯度如下: 0.00–0.10 min 与7% 流动相 B/93%a, 0.11–0.60 min 与65% 流动相 B/35%a, 0.61–2.10 分钟以95% 个移动的阶段 B/5%a, 2.11–4.00 min 以7% 个移动的阶段 B/93%a。 注: 计算额外列容积、仪器平台的容纳量以及 USP < 621 > 色谱准则16所述的分析物保留因子。</li>
</ol>7. 样本测量主文件
注: 在 "样本测量主文件"中, 指定患者样本, 并启动 HPLC-质谱/ms 分析, 并进行数据评估。生成两个独立的模板文件, 包括低质量和高质量的控制对;其中一个模板包括 qc 配对 A 和 C, 另一个是 qc 副 B 和 D。
<ol><li>创建新的 "示例测量主文件"。选择上面提到的 "ms 调谐文件"、"ms 文件"和"入口文件" (第6节), 将它们插入到每个示例行中, 并指定具有15µL 的注入量。</li>
	<li>按升序排列, 添加校准0–7和质量控制 (qc) 对 a/C 或 qc 对 B/D 的 "示例文本"。</li>
	<li>指定示例类型。为质量控制对选择校准和 "QC" 的示例类型 "标准"。</li>
	<li>指定相应校准和质量控制的每种抗生素物质的浓度 (见电子表格, 浓度 [µg/毫升] Cal 7–Cal 0, QC A/C 或 B/D,)。</li>
	<li>程序的 "数据评估方法"。使用在仪器优化过程中优化的转换 (第5节)。将每种抗生素与相应的同位素标的标准 (如美罗培南6) 相匹配。</li>
</ol>8. 样品清理和高效液相色谱-ms/毫秒分析
注: 对于每个样品批次, 处理和分析一组具有低和高抗生素浓度的配对质量控制 (qc a/C 或 qc B/D)。在不同批次之间, 配对 QC 样品用在一个备用序列 (例如, 在1天, 选择 '样品测量主文件 '包括 qc 对 A/C; 在2天, 选择一个包括 qc 对 B/D。血清样品的处理如图 1所示。
<ol><li>准备沉淀剂10% 甲基叔丁基醚在甲醇 (10:90, v/v) (例如, 预先填充一个25毫升容积瓶与2.5 毫升的甲基叔丁基醚和填充它到25毫升与绝对甲醇)。</li>
	<li>将 C8 反转相置于柱腔内。将其连接到液相色谱和质谱仪的流向。</li>
	<li>生成示例列表。打开相应的 "样本测量主文件"模板, 并将要处理的病人样本添加到列表中。生成多达20个患者样本的组, 并向他们提供相应的质量控制对。</li>
	<li>HPCL 系统使用 "入口文件"控制软件: 将 "湿素数" 功能设置为50% 个移动相 A/50%b, 湿素数为2分钟, 流速为1毫升/分钟。</li>
	<li>刷新注射器。为此, 在控制软件中执行6冲程600µL。</li>
	<li>平衡 C8 反相柱。使用该软件, 打开 "输入文件"中的流, 并将其与7% 移动相 B/93%a 进行冲洗, 最小为5分钟, 使用流速为0.5 毫升/分钟. 验证柱温30°c。</li>
	<li>解冻患者样品, 一整除校准 0–7, 和质量控制对 (即 a/B 或 C/D)。</li>
	<li>用重复的吸管, 添加25µL 的内部标准混合到100µL 校验仪, QC 样品, 或患者血清在1.5 毫升聚丙烯管, 并涡管几秒钟。</li>
	<li>在台式振动筛的室温下孵育5分钟的混合物 (例如, 在1200转每分钟)。</li>
	<li>用重复的吸管, 添加150µL 的沉淀试剂的样本内部标准混合。</li>
	<li>再次, 涡管几秒钟, 并孵化它在室温下的台式振动筛 (例如, 在 1200 rpm) 5 分钟。</li>
	<li>离心机悬浮在 2万 x g在桌上离心机为10分钟在4°c。</li>
	<li>用高效液相色谱法稀释上清 1:3, 用微插入玻璃瓶, 将其作为加工样品装入 autosampler。</li>
	<li>在 "样本测量控制文件"中手动启动高效液相色谱-ms/毫秒分析。 注: 对于长时间存储, 请根据制造商的建议彻底冲洗分析柱 [例如, 0.5 毫升/分甲醇-水 (50:50, v/v)] 以防止相崩。</li>
</ol><img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58148/58148fig1.jpg"> 图 1: 示例清理的示意图表示形式.高离心力下的蛋白质沉淀给出致密颗粒和清液, 表明蛋白质沉淀是完全的。整个处理时间约为30分钟, 包括样品清理、色谱分离和 ms/毫秒分析。<a href="https://www.jove.com/files/ftp_upload/58148/58148fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
9. 质量评估和量化
<ol><li>要处理样本, 请打开相应的 "样本测量控制文件", 选择校准程序、质量控制和患者样本, 并用 "抗生素量化方法"对其进行评估。
</li>
	<li>检查特定分析物的峰值是否正确集成。检查每个校准器、QC 和病人样本的峰值, 并在必要时手动将其重新整合到基线。</li>
	<li>研究校准曲线并检查它是否满足以下质量标准: a) 线性度在整个校准范围内, b) 校准系数 r2 > 0.995, c) 每一个校准标准的偏差在15% 的标称值, 除了量化下限 (LLOQ), 其中20% 是必需的。</li>
	<li>拒绝符合上述标准的校准标准, 并重新评估校准曲线, 包括回归分析。</li>
	<li>研究质量控制, 检查偏差是否在标称值的15% 以内。</li>
	<li>如果病人的浓度超过最高校验仪的浓度, 稀释样品与蒸馏水, 高达 1:5 (例如, 100 µL 的血清加400µL 蒸馏水) 在样品清理之前。Reperform 步骤8.8–8.14 该特定的示例并重新处理它。</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+pharmacokinetic/pharmacodynamic+principles+in+critically+ill+patients:+optimizing+efficacy+and+reducing+resistance+development.">Applying pharmacokinetic/pharmacodynamic principles in critically ill patients: optimizing efficacy and reducing resistance development.</a> Seminars in Respiratory and Critical Care Medicine. 36 (1), 136-153 (2015).</li><li>Imani, S., Buscher, H., Marriott, D., Gentili, S., Sandaradura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+a+retrospective+study+of+beta-lactam+concentration-toxicity+relationships.">Too much of a good thing: a retrospective study of beta-lactam concentration-toxicity relationships.</a> Journal of Antimicrobial Chemotherapy. 72 (10), 2891-2897 (2017).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> Pharmacy &amp; Therapeutics. 40 (4), 277-283 (2015).</li><li>Pulcini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+stewardship:+update+and+perspectives.">Antibiotic stewardship: update and perspectives.</a> Clinical Microbiology and Infection. 23 (11), 791-792 (2017).</li><li>Cairns, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+a+multidisciplinary+antimicrobial+stewardship+team+on+the+timeliness+of+antimicrobial+therapy+in+patients+with+positive+blood+cultures:+a+randomized+controlled+trial.">The impact of a multidisciplinary antimicrobial stewardship team on the timeliness of antimicrobial therapy in patients with positive blood cultures: a randomized controlled trial.</a> Journal of Antimicrobial Chemotherapy. 71 (11), 3276-3283 (2016).</li><li>Baur, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+stewardship+on+the+incidence+of+infection+and+colonisation+with+antibiotic-resistant+bacteria+and+Clostridium+difficile+infection:+a+systematic+review+and+meta-analysis.">Effect of antibiotic stewardship on the incidence of infection and colonisation with antibiotic-resistant bacteria and Clostridium difficile infection: a systematic review and meta-analysis.</a> Lancet Infectious Diseases. 17 (9), 990-1001 (2017).</li><li>Roberts, J. A., Norris, R., Paterson, D. L., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+drug+monitoring+of+antimicrobials.">Therapeutic drug monitoring of antimicrobials.</a> British Journal of Clinical Pharmacology. 73 (1), 27-36 (2012).</li><li>Paal, M., Zoller, M., Schuster, C., Vogeser, M., Schutze, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+cefepime,+meropenem,+ciprofloxacin,+moxifloxacin,+linezolid+and+piperacillin+in+human+serum+using+an+isotope-dilution+HPLC-MS/MS+method.">Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC-MS/MS method.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 102-110 (2018).</li><li>Vogeser, M., Seger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+associated+with+the+use+of+liquid+chromatography-tandem+mass+spectrometry+in+the+clinical+laboratory.">Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory.</a> Clinical Chemistry. 56 (8), 1234-1244 (2010).</li><li>United States Pharmacopeia and National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+&#60;621&#62;.">Chapter &#60;621&#62;.</a> CHROMATOGRAPHY (USP 37-NF 32 S1). , 6376-6385 (2014).</li><li>Wong, G., Sime, F. B., Lipman, J., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+we+use+therapeutic+drug+monitoring+to+improve+outcomes+from+severe+infections+in+critically+ill+patients?.">How do we use therapeutic drug monitoring to improve outcomes from severe infections in critically ill patients?.</a> BMC Infectious Diseases. 14, 288 (2014).</li><li>. Cefepime hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf</a> (2016)</li><li>. Meropenem: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf</a> (2006)</li><li>. Ciprofloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf</a> (2016)</li><li>. Moxifloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf</a> (2010)</li><li>. Linezolid: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf</a> (2011)</li><li>. Piperacillin and Tazobactam: Highlighs of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf</a> (2017)</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+biobanking+conditions+on+six+antibiotic+substances+in+human+serum+assessed+by+a+novel+evaluation+protocol.">Effects of biobanking conditions on six antibiotic substances in human serum assessed by a novel evaluation protocol.</a> Clinical Chemistry and Laboratory. 54 (2), 265-274 (2016).</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+piperacillin,+tazobactam,+cefepime,+meropenem,+ciprofloxacin+and+linezolid+in+serum+using+an+isotope+dilution+UHPLC-MS/MS+method+with+semi-automated+sample+preparation.">Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.</a> Clinical Chemistry and Laboratory. 53 (5), 781-791 (2015).</li></ol>

作者没有什么可透露的。

在本手稿中, 我们报告了一个简单和稳健的串联质谱法的方法, 以量化的常用抗生素在 ICU19, 即头孢吡肟, 美罗培南, 环丙沙星, 莫西沙星, 利奈, 和哌拉西林14。电子表格伴随着编写抗生素库存解决方案、校准仪和质量控制的手稿, 同时考虑到抗生素的纯度和 counterions 的分子量。鉴于抗生素浓度相当高, 从分析的角度来看, 它们的量化应该不是特别的挑战。因此, 我?...

尽管抗生素已经彻底改变了医学的实践, 但严重的细菌感染仍然是导致严重疾病发病率和死亡率的主要原因1。在这方面, 迅速管理适当剂量的适当抗感染, 对疾病控制2至关重要。
越来越多的证据表明, 用广谱抗生素进行的经验治疗随着患者数量的复杂性越来越成问题。对于重症监护病房 (ICU) 尤其如此, 在这种情况下, 关键药动学 (PK) 参数的巨大的个体间变异性经常被观察到3,4。因此, ICU 患者即将面临亚治疗水平的危险, 治疗成功率不足5,6。再次, 患者不必要地暴露在过高的抗生素浓度, 可能导致严重的不良事件, 没有临床好处7。抗生素滥用和剂量不足也助长了抗生素耐药性的传播, 这正日益成为对公共卫生8的威胁。
为了改进抗生素的使用, 并尽可能长时间地保存 effectivenessas, 世界卫生组织于2015年发起了一项关于抗菌素耐药性的全球行动计划.抗生素管理计划是10国家公共卫生战略中谨慎使用抗菌药物的一个重要基石, 帮助临床医生提高患者护理质量11 , 同时显著降低抗生素耐药性12。通过应用治疗药物监测 (TDM) 在个别患者中的抗生素剂量是这方面的一个关键工具13。
到目前为止, 商业上可用的 TDM 化验仅可用于糖肽类抗生素和氨基糖苷类。对其他类中的物质进行量化通常需要内部方法开发或验证, 这可能很麻烦。因此, 我们详细介绍了一种基于稳健质谱分析的协议, 该方法可用于在其临床相关浓度范围内对 ICU 中最相关的抗生素进行量化14。该方法最近在我们的质谱仪中建立, 并已被应用于 ICU 的常规 TDM 从那以后。该程序使用一个简单的分析设置和统一的样本清理, 允许快速实施抗生素 TDM 在许多设施的质谱能力。
本文所描述的协议是针对人血清中头孢吡肟、美罗培南、环丙沙星、莫西沙星、利奈和哌拉西林的定量化而优化的, 采用同位素稀释液相色谱 (LC) 与串联质量光谱 (ms/毫秒)。对于同位素稀释 LC-ms/毫秒方法, 稳定的同位素标记化合物添加到一个特定的矩阵 (如血清) 感兴趣的样本。同位素标记的标准可以区别于它们未标记的对应物, 即由于天然分子的不同分子量和它们的破碎产物, 而被称为母体离子对女儿离子的转变。由于同位素标记化合物与未标记的对应物具有几乎相同的整体物理化学行为, 因此它们是 ms/ms 的理想内部标准, 允许近基质独立的分析物质量化, 具有高度的精确度15。目前, 许多可用于小分子量化的稳定同位素标记内部标准, 包括抗菌素的 TDM, 都是商业上可用的。
用分析 C8 烷基链长度反相柱 (100 毫米 x 2.1 毫米, 3 µm 粒度), 对所述协议中的抗生素分析仪进行了色谱分离。在方法开发过程中, 所有分析物的内部标准归一化矩阵因子介于94.6% 和105.4% 之间, ≤8.3% 的变化系数为14。

<table>
	<tbody>
		<tr>
			<td>cefepime hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>1097636</td>
			<td>USP Reference Standard</td>
		</tr>
		<tr>
			<td>meropenem trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001252</td>
			<td>EP Reference Standard</td>
		</tr>
		<tr>
			<td>ciprofloxacin</td>
			<td>Sigma-Aldrich</td>
			<td>17850</td>
			<td></td>
		</tr>
		<tr>
			<td>moxifloxacin hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>SML1581</td>
			<td></td>
		</tr>
		<tr>
			<td>linezolid</td>
			<td>Toronto Research Chemicals</td>
			<td>L466500</td>
			<td></td>
		</tr>
		<tr>
			<td>piperacillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>93129</td>
			<td></td>
		</tr>
		<tr>
			<td>cefepime-13C12D3 sulfate</td>
			<td>Alsachim</td>
			<td>C1297</td>
			<td>Isotope labelled internal standard for cefepime</td>
		</tr>
		<tr>
			<td>meropenem-D6</td>
			<td>Toronto Research Chemicals</td>
			<td>M225617</td>
			<td>Isotope labelled internal standard for meropenem</td>
		</tr>
		<tr>
			<td>ciprofloxacin-D8</td>
			<td>Toronto Research Chemicals</td>
			<td>C482501</td>
			<td>Isotope labelled internal standard for ciprofloxacin</td>
		</tr>
		<tr>
			<td>moxifloxacin-13C1D3 hydrochloride</td>
			<td>Toronto Research Chemicals</td>
			<td>M745003</td>
			<td>Isotope labelled internal standard for moxifloxacin</td>
		</tr>
		<tr>
			<td>linezolid-D3</td>
			<td>Toronto Research Chemicals</td>
			<td>L466502</td>
			<td>Isotope labelled internal standard for linezolid</td>
		</tr>
		<tr>
			<td>piperacillin-D5</td>
			<td>Toronto Research Chemicals</td>
			<td>P479952</td>
			<td>Isotope labelled internal standard for piperacillin</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>JT Baker</td>
			<td>8402</td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC grade water</td>
			<td>JT Baker</td>
			<td>4218</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Biosolve</td>
			<td>6914132</td>
			<td></td>
		</tr>
		<tr>
			<td>acetic acid</td>
			<td>Biosolve</td>
			<td>1070501</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium formate</td>
			<td>Sigma-Aldrich</td>
			<td>70221-25G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl methyl ether</td>
			<td>Merck</td>
			<td>101845</td>
			<td></td>
		</tr>
		<tr>
			<td>Fortis 3 &#956;m C8 100 * 2.1 mm</td>
			<td>Fortis</td>
			<td>F08-020503</td>
			<td></td>
		</tr>
		<tr>
			<td>Ti-PEEK-encased Prifilter (2 &#956;m)</td>
			<td>Chromsystems</td>
			<td>15011</td>
			<td></td>
		</tr>
		<tr>
			<td>2795 Alliance HPLC system</td>
			<td>Waters</td>
			<td>176000491</td>
			<td></td>
		</tr>
		<tr>
			<td>Quattro micro API Tandem Quadrupole System</td>
			<td>Waters</td>
			<td>720000338</td>
			<td></td>
		</tr>
		<tr>
			<td>QuanLynx 4.1 software</td>
			<td>Waters</td>
			<td>/</td>
			<td>Data evaluation software provided by the mass spectrometer manufacturer</td>
		</tr>
	</tbody>
</table>

multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses

许多临床设施对抗生素的治疗药物的需求不断增加, 特别是在实施医院抗生素管理计划方面。
在目前的工作中, 我们提出了一个多用途的高效液相色谱-串联质谱 (HPCL ms/ms) 协议, 以量化的头孢吡肟, 美罗培南, 环丙沙星, 莫西沙星, 利奈, 和哌拉西林, 常用重症监护病房的抗生素。该方法以前是根据欧洲药物管理局的指导方针进行全面验证的。
经过快速的样品清理, 在4分钟内将分析物分离在 C8 反相色谱柱上, 用电喷雾电离 (ESI +) 质谱中相应的稳定同位素标记的内部标准进行多重反应。时间监视 (MRM)。所提出的方法采用简单的仪器设置, 色谱条件均匀, 可用于临床实验室的日常和强健的抗生素治疗药物监测。校准曲线横跨药代动力学的浓度范围, 从而包括抗生素量接近最小抑制浓度 (MIC) 的敏感细菌和峰值浓度 (C最大), 得到的丸管理方案。如果不需要在样品清理前进行血清稀释, 则可以通过多种测量方法获得被管理的抗生素曲线下的区域。

使用描述的协议, 在图 2中描述了一个典型的色谱。根据美国药典 (USP) 色谱准则16, 目前系统中的柱死体积是用 ~ 0.22 毫升和额外柱容积 (包括喷油器、油管和连接器) 确定的, 其数量为0.08 毫升, 给0.30 毫升的容纳量。计算出的所有分析物的保留因子为 2.8 (用于头孢吡肟)-4.2 (用于哌拉西林)。
<p class="jove_content" fo:keep-together.within-page="1...

Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

同位素稀释-高效液相色谱-抗生素在危重症患者中的多重治疗药物监测

在此, 我们提出了一种基于串联质谱的协议, 用于定量治疗重症监护病房中的常用抗生素, 即头孢吡肟、美罗培南、环丙沙星、莫西沙星、利奈和哌拉西林。

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the ...

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12.8K 次观看.  LMU Munich. 在此, 我们提出了一种基于串联质谱的协议, 用于定量治疗重症监护病房中的常用抗生素, 即头孢吡肟、美罗培南、环丙沙星、莫西沙星、利奈和哌拉西林。

视频: Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

支气管Thermoplasty:严重哮喘的一种新的治疗方法

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

科研

医学

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

在体测量小鼠肺血管内皮表面层

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load &#62;1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads &#60;1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.

Drug resistance testing for HIV-1 infected individuals failing antiretroviral therapy (ART) can guide future therapies and improve treatment outcomes. Optimizing individual and population health outcomes in high HIV prevalence but resource-limited settings will ultimately require affordable and accessible drug resistance genotyping and interpretation methods.

对于资源有限设置一个负担得起的HIV-1耐药性监测方法

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

一个简单的临界尺寸股骨缺损模型小鼠

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring

The use of ultra-high field MRI as a non-invasive way to obtain phenotypic information of rodent models for polycystic kidney disease and to monitor interventions is described. Compared with the traditional histological approach, MRI images can be acquired in vivo, allowing for longitudinal follow-up.

在多囊肾小型啮齿动物模型采用超高场磁共振成像<em&gt;在体内</em&gt;表型和药物监测

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

在小鼠节段性股临界尺寸缺陷模型的建立接骨板稳定

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Standardized Hemorrhagic Shock Induction Guided by Cerebral Oximetry and Extended Hemodynamic Monitoring in Pigs

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an insufficient oxygen supply and irreversible organ failure. The brain exerts only limited compensation capacities and is particularly at high risk of severe hypoxic damage.This article demonstrates the reproducible induction of life-threatening hemorrhagic shock in a porcine model by means of calculated blood withdrawal. We titrate shock induction guided by near-infrared spectroscopy&#160;and extended hemodynamic monitoring to display systemic circulatory failure, as well as cerebral microcirculatory oxygen depletion. In comparison to similar models that primarily focus on predefined removal volumes for shock induction, this approach highlights a titration by means of the resulting failure of macro- and microcirculation.

Hemorrhagic shock is a severe complication in seriously injured patients, which leads to life-threatening oxygen undersupply. We present a standardized method to induce hemorrhagic shock via blood withdrawal in pigs that is guided by hemodynamics and microcirculatory cerebral oxygenation.

由脑氧测量和猪期扩展血液动力学监测指导的标准化出血性休克诱导

Hemorrhagic shock ranks among the main reasons for severe injury-related death. The loss of circulatory volume and oxygen carriers can lead to an ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

大鼠胡萝卜动脉压力控制段气球损伤与腹腔ventit 治疗应用

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

经过验证的LC-MS/MS面板，用于在小头发样本中量化11种耐药结核病药物

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...