Name: multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses
Uploaded: 2018-08-30T15:00:00.000+00:00
Duration: 11 min 17 s
Description: Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

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>

<ol>
	<li>Fleischmann, C., 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=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Dellinger, R. P., 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=Surviving+Sepsis+Campaign:+international+guidelines+for+management+of+severe+sepsis+and+septic+shock,+2012.">Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012.</a> Intensive Care Medicine. 39 (2), 165-228 (2013).</li><li>Lodise, T. P., Drusano, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics:+optimal+antimicrobial+therapy+in+the+intensive+care+unit.">Pharmacokinetics and pharmacodynamics: optimal antimicrobial therapy in the intensive care unit.</a> Critical Care Clinics. 27 (1), 1-18 (2011).</li><li>Macedo, R. S., Onita, J. H., Wille, M. P., Furtado, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics+of+antimicrobial+drugs+in+intensive+care+unit+patients.">Pharmacokinetics and pharmacodynamics of antimicrobial drugs in intensive care unit patients.</a> Shock. 39, 24-28 (2013).</li><li>Petersson, J., Giske, C. G., Eliasson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+dosing+of+piperacillin+and+meropenem+fail+to+achieve+adequate+plasma+concentrations+in+ICU+patients.">Standard dosing of piperacillin and meropenem fail to achieve adequate plasma concentrations in ICU patients.</a> Acta Anaesthesiologica Scandinavica. 60 (10), 1425-1436 (2016).</li><li>Abdul-Aziz, M. H., Lipman, J., Mouton, J. W., Hope, W. W., 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=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 &amp;mu;m C8 100 * 2.1 mm</td><td>Fortis</td><td>F08-020503</td><td></td></tr><tr><td>Ti-PEEK-encased Prifilter (2 &amp;mu;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><tr><td>Supplemental File</td><td></td><td></td><td>Stock Solutions</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 ...

multiplex-therapeutic-drug-monitoring-isotope-dilution-hplc-msms

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JoVE Journal

Medicine

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

科研

化学

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

癌症研究

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

生物化学

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

医学

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

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. ...

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in seconds, which leads to accumulation of levels that approach or exceed those of GTP pools. Stress reversal occasions a rapid disappearance of (p)ppGpp, often with a half-life of less than a minute. The presence of (p)ppGpp results in alterations of cellular gene expression and metabolism that counter the damaging effects of stress. Gram-negative and Gram-positive bacteria have different response mechanisms, but both depend on (p)ppGpp concentration. In any event, there is a need to simultaneously monitor many radiolabeled bacterial cultures at time intervals that may vary from 10 seconds to hours during critical stress transition periods. This protocol addresses this technical challenge. The method takes advantage of temperature- and shaker-controlled microtiter dish incubators that allow parallel monitoring of growth (absorbance) and rapid sampling of uniformly phosphate-radiolabeled cultures to resolve and quantitate nucleotide pools by thin-layer chromatography on PEI-cellulose. Small amounts of sample are needed for multiple technical and biological replicates of analyses. Complex growth transitions, such as diauxic growth and rapid (p)ppGpp turnover rates can be quantitatively assessed by this method.

Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli pppGpp Followed by Thin Layer Chromatography

The growth of radiolabeled bacterial cultures in microtiter dishes facilitates high throughput sampling that allows multiple technical and biological replicate assays of nucleotide pool abundance, including that of (p)ppGpp. The effects of growth transitions provoked by sources of physiological stress as well as recovery from stress can be monitored.

使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱

The (p)ppGpp nucleotide functions as a global regulator in bacteria in response to a variety of physical and nutritional stress. It has a rapid onset, in ...

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

摘要

探索更多视频

此视频中的章节

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

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

使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱

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

摘要

探索更多视频

此视频中的章节

不相关的代谢组学，生物源使用超高效液相色谱 - 高分辨质谱（UPLC-HRMS）

纳米颗粒对荷瘤小鼠药相结合的样品提取及同时色谱定量分析

增强组织样品使用组合前体同位素标记和标记等压的复用（cPILOT）

液相色谱离子迁移 QTOF 质谱测定蛋白质生物中宿主细胞蛋白的 HS-MRM 法

采用多段注射-毛细管电泳质谱法进行高通量和综合药物监测

使用组合前体同位素标签和同位素标记 （cPILOT） 实现自动样品多路复用

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

激光捕获显微切割液相色谱质谱 (LCM-LC/MS) 对肺结核患者药物的空间量化

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

使用微蒂特萝卜无线电标签进行多维欧大肠杆菌测量 (p) ppGpp,然后是薄层色谱

使用组合前体同位素标签和同位素标记（cPILOT）实现自动样品多路复用