. 和电视得到国立卫生研究院 ( R01 AI111965 ) 国家反应和传染性疾病研究所 ( NIAID ) 的支持。内容完全是作者的责任, 并不一定代表国家过敏和传染性疾病研究所或国立卫生研究院的官方观点。MC 是澳大利亚澳洲的主要研究员。. 是澳大利亚国家卫生与医学研究委员会 (澳洲) 高级研究员, 而电视是澳大利亚澳洲行业职业发展2级研究员。杨永强 S 是一个任命的年轻大使2017为 ASM (美国微生物学协会) 并且由澳洲研究生奖支持。 这项工作的一部分是在 12th Australiasian 会议上囊性纤维化在墨尔本 (5-8th 2017年8月)。

对道德的认可是由蒙纳士大学人类研究伦理委员会 (MUHREC) 获得的. 
 1. 分析的应用: 患者样本收集 <ol> <li> 记录患者服用标准剂量的150毫克 ivacaftor 或 ivacaftor 125 毫克/lumacaftor 200 毫克的时间。
	<li> 记下病人血样采集的确切时间. 
	注: 我们建议在24小时的课程中收集4-5 样本。如果只有一个样品的集合是可能的, 被表明的时间点是 2.5-4 h 岗位 ivacaftor 或 ivacaftor-lumacaftor 组合药量作为 C 最大 集中在稳定状态将是可实现的在和 #62 以后; 5 天连续治疗. </li>
	<li> 收集4-5 毫升的血液在商业上可用未经处理的蓝色管. </li>
	<li> 在收集完整个血液后, 让血液凝结, 使其在室温下不受干扰. 
	注意: 这通常需要15-30 分钟. </li>
	<li> 离心在1,000-2、000 x g 处将血块移除, 在4和 #176 中为10分钟; C 在冷冻离心机中。将产生的上清液指定为等离子体. </li>
	<li> 在离心后, 立即将上清 (等离子) 转化为使用巴斯德吸管的清洁聚丙烯管。用患者 ID ( 例如 , 患者 1), 使用药物 ( 例如 , ivacaftor-lumacaftor 组合) 和病人样本收集时间 ( 如 , 2.5 h post-dosage) 来标记血浆管. </li>
	<li> 在处理过程中维护2-8 和 #176 的样品;处理完成后, 将样品存放在 #8211; 20 和 #176; C. </li>
	<li> 如有必要, 请将经批准的信使的样品运送到分析研究所. 
	注: 样品要在干冰运到到达时, 并应保存在和 #8211; 80 和 #176; C 直到分析. </li>
</ol> 2。发生的样品和标准的准备和处理 注意: 来自健康献血者的血浆和 #239; ve ivacaftor/lumacaftor 治疗是从澳大利亚红十字会获得的。为确保产品的完整性, 所有样品/标准应保持在2-8 和 #176; C 在收集和处理过程中。处理完成后, 将样品存放在 #8211; 20 和 #176; C. 
<ol> <li> 称其为内部标准的参考, 并对其进行权衡, 并对其进行 LUMA。
	<li> 在100和 #181 中, 为每种分析物 (M1、M6 和 LUMA) 分别准备两个独立的库存解决方案: LC-MS 级甲醇; g/毫升; 10 和 #181; g/毫升. 
	注: 化合物将在台式10天后过期. </li>
	<li> 在每个分析运行之前, 通过稀释校准标准的库存解决方案, 以实现以下浓度: 0.01、0.025、0.05、0.1、0.2、0.5、1.0、2.5、5.0 和10.0、#181,这些示例被称为标准. </li>
	<li> 沉淀蛋白质, 准备0.1% 甲酸 (FA) 在乙腈 (ACN, LC 级), 并留在冰箱, 直到需要. </li>
</ol> 3。已发生的样品和标准的预处理 <ol> <li> 允许患者和空白血浆样品平衡到室温. </li>
	<li> 涡旋将每个样品的每个等离子体样本混合到十五年代. </li>
	<li> 传输100和 #181; 将空白等离子体分为标准或患者样品, 用于分析1.5 毫升聚丙烯离心管. </li>
	<li> 在每个标准管中添加内部标准的 "," ( 例如 , 0.01, 0.025, 0.05, 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 和 #181; g/毫升, 以涵盖潜在的浓度范围), 关闭盖子, 和涡流 10.0 s. 注意, 内部标准仅添加到空白血浆和没有内部标准被增加到患者样品. </li>
	<li> 重复步骤 3.4. 其他内部标准 M1、M6 和 LUMA. </li>
	<li> 简单地向下旋转每个管, 以确保盖子上没有液滴. </li>
	<li> 添加200和 #181; 0.1% FA 的混合 ACN 到每管中沉淀血浆蛋白. </li>
	<li> 在大约十五年代的时候强烈地漩涡混合物. </li>
	<li> 允许管子在冰箱中站立10分钟. </li>
	<li> 离心 1万 x g 为10分钟, 在4和 #176; C, 如果可能的话. </li>
	<li> 筛选200和 #181; 通过 13 mm 注射器过滤器将上清液分为1.5 毫升 HPLC 瓶. </li>
	<li> 将100和 #181; 将上清的 L 转化为 LC-ms 瓶, 进行 HPLC 分析. </li>
</ol> 4。高效液相色谱-ms 分析 注意: 在 LC-ms 系统上进行了高效液相色谱分析, 并结合三极质谱仪 ( 表 1 )。
<ol> <li> 打开 LC-MS 系统, 将纸盒与样中的示例一起放置. </li>
	<li> 将列附加到保护列并连接到 LC-MS 系统. </li>
	<li> 附加两个流动相瓶 (瓶 A: 100% ACN; 瓶子 B: 水中的0.1% 甲酸) 和平衡的 LC-MS 系统. </li>
	<li> 将下列参数合并到 LC-MS 协议中. 
	<ol> <li> 在以2:1 的比率 (废物: MS 入口) 进入质谱仪之前, 拆分流动相流. </li>
		<li> 使用在水中包含 100% ACN 和0.1% 甲酸的流动相, 在0.5 毫升/分钟的流速下执行梯度洗脱 (起始点在40:60、v/v)。请注意, 在整个分析过程中, 移动阶段的体积百分比会发生变化. </li>
		<li> 以正电喷雾电离模式操作质谱仪. </li>
		<li> 确保 LC-MS 设置如下: 离子喷雾电压 4.5 kV, 碰撞能量295.9 伏, 雾化气体: 氮气在3升/分; 碰撞气体: 氩气; 干燥气体流量20升/分, 透镜电压 Q3:-22 V, 去温度250和 #176; C 带热块温度为400和 #176; C. </li>
		<li> 注入10和 #181; 每个样本的 L 体积. </li>
		<li> 使用多反应监测 (MRM) 检测分析。监视 m/z 392.49 和 #8594 的离子跃迁; 393, m/z 408.49 和 #8594; 409, m/z 422.47 和 #8594; 423 和 m/z 452.40 和 #8594; 分别为 IVA-M1、IVA-M6 和 LUMA. 
		注意: 根据 FDA 的标准, 新的优化方法尚未完全验证. </li>
	</ol> </li> </ol> 5。校准曲线 <ol> <li> 在每个分析运行前构造 LC-MS 校准曲线, 使用四分析的峰值面积比值与内部标准之间的关系, 以及校准标准的标称浓度ivacaftor (IVA-M1、IVA-M6 或 LUMA) 在 LC-MS 设置程序中. 
	注: 校准曲线构造的精确步骤取决于所使用的 LC-MS 设备的模型。此信息可在设备手册中获得. </li>
	<li> 根据浓度的倒数, 通过加权 1/C 执行线性最小二乘法回归分析. </li>
</ol>

<ol>
	<li>Schneider, E. K., 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=Drug-drug+plasma+protein+binding+interactions+of+ivacaftor.">Drug-drug plasma protein binding interactions of ivacaftor.</a> J Mol Recognit. 28 (6), 339-348 (2015).</li><li>Solomon, M., Leatte, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Treatments for Cystic fibrosis. , (2009).</li><li>O'Sullivan, B. P., Flume, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+approach+to+lung+disease+in+patients+with+cystic+fibrosis.">The clinical approach to lung disease in patients with cystic fibrosis.</a> Semin Respir Crit Care Med. 30 (5), 505-513 (2009).</li><li>Ramsey, B. W., 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=A+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation.">A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.</a> N Engl J Med. 365 (18), 1663-1672 (2011).</li><li>Wainwright, C. E., 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=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. 373 (3), 220-231 (2015).</li><li>Hadida, S., 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=Discovery+of+N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide+(VX-770,+ivacaftor),+a+potent+and+orally+bioavailable+CFTR+potentiator.">Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator.</a> J Med Chem. 57 (23), 9776-9795 (2014).</li><li>FDA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FDA Advisory Commitee Briefing Material VERTEX-FDA Pulmonary-Allergy drugs advisory commitee. 98 , (2015).</li><li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=False+dawn+for+cystic+fibrosis+disease+modifiers?.">False dawn for cystic fibrosis disease modifiers?.</a> Nat Rev Drug Discov. 13 (10), 713-714 (2014).</li><li>Veit, G., 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=Some+gating+potentiators,+including+VX-770,+diminish+DeltaF508-CFTR+functional+expression.">Some gating potentiators, including VX-770, diminish DeltaF508-CFTR functional expression.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>Cholon, D. M., 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=Potentiator+ivacaftor+abrogates+pharmacological+correction+of+DeltaF508+CFTR+in+cystic+fibrosis.">Potentiator ivacaftor abrogates pharmacological correction of DeltaF508 CFTR in cystic fibrosis.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>EMA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment report ORKAMBI (ivacaftor/lumacaftor) European medicines agency. , (2015).</li><li>VERTEX. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertex prescribing infomation. , (2015).</li><li>Matthes, E., 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=Low+free+drug+concentration+prevents+inhibition+of+F508del+CFTR+functional+expression+by+the+potentiator+VX-770+(ivacaftor).">Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).</a> Br J Pharmacol. 173 (3), 459-470 (2016).</li><li>Schneider, E. K., 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=Development+of+HPLC+and+LC-MS/MS+methods+for+the+analysis+of+ivacaftor,+its+major+metabolites+and+lumacaftor+in+plasma+and+sputum+of+cystic+fibrosis+patients+treated+with+ORKAMBI+or+KALYDECO.">Development of HPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with ORKAMBI or KALYDECO.</a> J Chrom B Analyt Technol Biomed Life Sci. 1038, 57-62 (2016).</li><li>Su, Q., 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=An+LC-MS/MS+method+for+the+quantitation+of+cabozantinib+in+rat+plasma:+application+to+a+pharmacokinetic+study.">An LC-MS/MS method for the quantitation of cabozantinib in rat plasma: application to a pharmacokinetic study.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 985, 119-123 (2015).</li></ol>

作者没有什么可透露的。

如前所述, 我们的小组首次开发和验证了高效液相色谱和 LC-MS 法快速检测和定量 ivacaftor 及其主要代谢物羟甲基 M1 (活性) 和 M6 (不活泼);lumacaftor 在 CF 患者血浆和痰液中的浓度为14。本组报告的检测方法成功地用于定量测定 CF 患者的血浆和痰中 LUMA、IVA-M1、IVA-M6 的浓度, 采用150毫克/q12 h (拉丁语术语: quaque12 小时;每12小时一次) 或200毫克/q12 h LUMA-125 毫克/q12 h 综合体组合14。对于大量的患者样品的高通量分析, 我们通过使用较小孔径的反相色谱柱 C8 (2.6 µm; 100 Å; 50 x 2.1 mm2) 和梯度溶剂系统而不是电流来优化报告方法。等洗脱。这样可以将运行时间减少到每样6分钟。
这种可靠和新颖的方法提供了一个简单, 灵敏和快速的方法, 治疗药物监测 ivacaftor 和 ivacaftor-lumacaftor 结合在生物流体。鉴于需要发展接触反应关系, 以最大限度地提高药物功效, 并包含医疗保健费用, 需要开发和验证更敏感的工具, 以协助临床医生使用循证疗法和高通量分析技术为患者从 cf. 我们的小组以前报告了部份地被证实的 HPLC-MS 为同时分析 IVA-M1, IVA-M6, 和 LUMA 在血浆和痰在 cf 患者14。为分析接受 ivacaftor 或 ivacaftor-lumacaftor 结合的患者建立一个通用和易于使用的解析协议, 将有助于对未来药代动力学研究结果进行数据比较和解释。使用 LC-MS 技术测量生物体液中的 IVA-M1、IVA-M6 和 LUMA 可能是探索与治疗结果相关的接触反应关系的有力工具。
更一般地, ivacaftor 或 ivacaftor-lumacaftor 结合在生物体液中的 HPLC 分析可能为实施个体化的治疗策略提供了分子基础, 并可能证明有用的理由, 使昂贵的 ivacaftor 或 ivacaftor lumacaftor 组合疗法更具成本效益, 可能表明需要减少频繁剂量。将该分析方法应用于临床药物动力学/药效学研究中, 有机会进一步研究 ivacaftor 或 ivacaftor-lumacaftor 结合对大患者的药代动力学参数。集体.

囊性纤维化 (CF) 是一种常见的遗传性疾病, 涉及的外分泌粘液腺体的肺, 肝, 胰, 和肠道导致渐进性多衰竭, 如肺功能减退和胰不足1, 2,3。Ivacaftor (美国食品和药物管理局) 和欧洲药物管理局 (EMA) 批准囊性纤维化反式膜电导调节剂 (CFTR) 增药物, 其临床疗效显著G551D-CFTR 的小子集的 cf 患者肺功能改善的临床研究 [甘氨酸 (G) 在位置551中被天门冬氨酸 (D)] 义突变 (CF 种群的 4-5%)4,5。这种口服药物增加了 CFTR 通道开放, 从而增加了氯离子流和作用于主要缺陷, 导致的临床表现 CF4,6。不幸的是, 在更常见的纯合 F508del 突变的患者中, CFTR 单药的单一性是不有效的 [在帧删除的错误, 导致在位置508的苯丙氨酸 (F) 的损失, 这在50%CF 填充7,8。
最近, FDA 批准了与 CFTR 校正药物 lumacaftor 的结合。巧妙的策略, 结合 CFTR 校正器 (lumacaftor, LUMA), 这将 F508del-CFTR 到细胞表面的调制器 (伊娃) 加强 CFTR 通道活动, 有效地扩大治疗窗口的大多数 CF 人口5.关于这些药物是否能履行他们的诺言的问题仍然存在, 因为一些相互矛盾的报告已经出现, 对他们的临床功效产生了怀疑9,10。此外, 肺功能的改善只是适度的 (2.6-4% 的 ivacaftor-lumacaftor 组合) 相比, 成功地在 G551D 突变 (10.6-12.5%)8患者单药单一治疗。潜在的拮抗药物-药物相互作用可能限制 ivacaftor-lumacaftor 结合的临床疗效的 LUMA, 来自其不太理想的药代动力学属性7,11。在细胞色素 P450 酶 (CYP) 的作用下, 主要对活性代谢产物羟甲基--IVA-M1、M1) 和非活动形态 (IVA-M6, M6)7,12进行了广泛的代谢。CYP3A4 诱导 LUMA, 另一方面, 没有广泛的代谢和主要排泄不变的粪便11。由于 CYP3A4 诱导诱导细胞色素代谢, ivacaftor (CYP3A4 基质) 浓度可降低。此外, 两个 LUMA 都是非常疏水性分子和99% 绑定到血浆蛋白, 这极大地限制了自由 (有效) 药物浓度1,13。
总的来说, 这些因素可能聚集在一起, 限制了 ivacaftor-lumacaftor 结合的临床疗效。目前尚不清楚 ivacaftor-lumacaftor 组合的剂量方案是否达到最佳血浆浓度, 或者治疗阈值是否保持在8。目前, 关于 ivacaftor 或 ivacaftor lumacaftor 的药代动力学参数如峰值和稳态等离子体浓度的信息缺乏。鉴于 ivacaftor 和 lumacaftor 的新陈代谢, 对接触反应关系的监测是实现 ivacaftor 或 ivacaftor-lumacaftor 疗法最佳剂量方案的必要条件。我们的小组最近出版了第一个 HPLC/LC-MS 方法, 以监测的接触-反应关系的 LUMA 和14。目前还没有其他方法来测量 ivacaftor、其代谢物和 lumacaftor 的浓度。为了让更大的患者集体的高通量分析, 并大大减少分析时间, 我们组通过使用较小孔径的反相色谱柱和梯度溶剂系统优化了报告的方法,降低成本和运行时间。

<table><tbody><tr><td>IVA (Cat#S114)&amp;nbsp;</td><td>SelleckChem (USA).&amp;nbsp;</td><td></td><td></td></tr><tr><td>LUMA (Cat#S1565)&amp;nbsp;</td><td>SelleckChem (USA).&amp;nbsp;</td><td></td><td></td></tr><tr><td>IVA-carboxylate (Cat# 510242247CS)&amp;nbsp;</td><td>Clearsynth (Canada).&amp;nbsp;</td><td></td><td></td></tr><tr><td>hydroxymethyl-IVA (Cat# 510240849CS)&amp;nbsp;</td><td>Clearsynth (Canada).&amp;nbsp;</td><td></td><td></td></tr><tr><td>Methanol (MeOH, LC-MS grade),&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>acetonitrile (ACN, LC-MS grade)&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>formic acid (FA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>triple-quadrupole Shimadzu 8030 LC-MS&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Phenomenex Kinetex (2.6 &amp;micro;m C8 100 &amp;Aring;; 50 &amp;times; 2.1mm)</td><td></td><td></td><td></td></tr><tr><td>(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>1.5 mL polypropylene microcentrifuge tube (VWR).&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Eppendorf Centrifuge 5430</td><td></td><td></td><td></td></tr><tr><td>13-mm syringe filter (0.45 &amp;micro;m nylon, GRACE, USA)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>[Phenomenex VEREX, 9 mm, PP, 300 &amp;micro;L, PTFE/Silicone septa].&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

optimized lc-ms/ms method for the high-throughput analysis of clinical samples of ivacaftor, its major metabolites, and lumacaftor in biological fluids of cystic fibrosis patients

囊性纤维化膜传导调节剂 (CFTR) 的缺陷是囊肿纤维化 (CF) 的病因, 是一种具有生命危险的肺部表现的疾病。Ivacaftor (Ivacaftor) 和 lumacaftor (LUMA) 结合是两种新的突破性药物, 直接调节有缺陷的 CFTR 蛋白的活性和贩运。然而, 对药代动力学/药效学参数和 ivacaftor 和 lumacaftor 的药理研究仍然缺乏认识。高效液相色谱-MS 技术同时分析接受标准 ivacaftor 或 ivacaftor-lumacaftor 结合的 ivacaftor、羟甲基 ivacaftor、ivacaftor 羧酸和 lumacaftor 在生物体液中的浓度治疗之前, 我们的小组和部分验证的 FDA 标准。然而, 为了允许高通量分析更多的患者样本, 我们组通过使用较小孔径的反相色谱柱 (2.6 µm、C8 100 Å; 50 x 2.1 mm) 和梯度溶剂系统对报告的方法进行了优化 (0-1 分钟: 40% B;1-2 分钟: 40-70% B;2-2. 7 分钟: 举行在 70% B;2.7-2.8 分: 70-90% B;2.8-4.0 分钟: 90% B 洗涤;4.0-4.1 分: 90-40% B;4.1-6.0 分钟: 举行在 40% B) 而不是一个等洗脱。本研究的目的是将每样样品的高效液相色谱-MS 分析时间从 ~ 15 分钟减少到每样6分钟, 这对于分析大量患者样本是必不可少的。这种权宜之计的方法将有相当大的效用, 研究的接触反应的关系, 这些突破 CF 药物。

我们最近报告了一种方法, 部分验证了 FDA 的标准, 在三重四极 LC MS 和 HPLC 检测系统, 使用 C8 柱 (5 µm, 3.9 mm x 50 mm 身份) 与流动相组成的 100% ACN 和0.1% 甲酸在水中 (40:60, v/v) 的流速为1毫升/分钟。在人类血浆中, 所有代谢物、ivacaftor、Iva-M1、Iva-M6 和 lumacaftor14的浓度范围从0.01 到10µg/毫升都观察到峰值的线性相关性。此方法已经过优化, 使 LC 保留时间大大减少, 因此, 该检测的完整运行时间对大量临床样品的高通量分析至关重要。通过使用较小孔径的反相色谱柱和梯度溶剂体系, 而不是等洗脱, 将每样样品的时间从 ~ 15 分钟缩短到仅6分钟, 从而优化了该检测。采用多反应监测 (MRM) 确定了分析的电喷雾阳性模式。此过程应在使用前 in-house 验证, 如我们先前详细介绍的14。
该方法的精确度为94.2% ±4.53% 到97.5% ± 2.94%, 具体取决于分析物14。校准范围从0.01 到10µg/毫升是适用于临床设置, 特别是鉴于在 ivacaftor lumacaftor 治疗, 非常低浓度的活性和 M1 的报告, 我们的组先前, 在血浆可能由于CYP3A4 的诱导 LUMA, 导致大面积的代谢紊乱14。对每种分析物的内准确度进行评估, 并在同一天在浓度为0.05、0.5 和8µg/毫升的六个独立准备的质量控制 (QC) 样品。对 inter-day 的准确度进行了评估, 六个独立准备的 QC 样品连续三天。通过相对标准偏差 (RSD) 计算精度和精度。对于每个 QC 示例, RSD 值应小于 15%15。所有四分析显示 RSD 值小于 15%14。检测下限 (lod) 和量化 (LOQ) 已建立: 对于 2.50 x 10-3 µg/ml 和 LOQ 7.57 x 10-3 µg/ml;对于 M1, LOD 4.57 x 10-4 µg/ml 和 LOQ 1.38 x 10-3 µg/ml;对于 M6 LOD 5.86 x 10-4 µg/ml 和 LOQ 1.78 x 10-3 µg/ml;对于 LUMA LOD 是 6.08 x 10-4 µg/毫升和 LOQ 1.84 x 10-3 µg/ml14。典型的色谱显示在图 1中。利用 100% ACN 和0.1% 甲酸在水中的流动相 (起始点在40:60、v/v) 上, 通过 C8 柱上的梯度洗脱 (2.6 µm; 100 Å; 50 mm x 2.1 mm) 获得了优化的色谱分辨率。采用梯度分离法, 将40% 的移动相 B 从0到1分钟;然后 40%-70% 的移动阶段 B 从1分钟到2分钟;持有70% 的移动阶段 B 从2分钟到2.7 分钟;从70% 到90% 的移动阶段 B 从2.7 分钟增加到2.8 分钟;持有90% 的移动阶段 B 从2.8 分钟到4.0 分钟的洗涤目的;最后从90% 到40% 的移动 B 从4.0 分钟返回到4.1 分钟;然后持有40% 的移动阶段 B 从4.1 分钟到6.0 分钟的初始条件 (图 1)。LC 保留时间如下 M6 1.0 分钟;为 M1 1.3 分钟;为: 1.55 分钟;对于 LUMA: 2.3 分钟 (图 1)。在所有的空白等离子体样品中, 均未观察到任何一个分析的保留时间, 也没有从 ivacaftor 与 lumacaftor 的结合中检测到干扰。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56084/56084fig1.jpg"> 
图 1:用10µg IVA-M6、IVA-M1、lumacaftor 和 ivacaftor 与泵的梯度洗脱相关联的人血浆的代表 LC-MS 色谱.0.1% 甲酸在水和泵 B 100% 乙腈 (0-1 分: 40% b; 1-2 分钟: 40%-70% b; 2-2. 7 分钟: 举行在 70% b; 2.7-2.8 分钟: 70%-90% b; 3.8-4.0 min: 90% b 洗涤; 4.0-4.1 min: 90%-40% b; 4.1 分钟-6.0 分钟: 40% 到分。m/z 392.49 →393的离子转换, m/ z 408.49 → 409, m/ z 422.47 →423和m/z 452.40 →453分别用于 ivacaftor、M1、IVA-M6 和 lumacaftor 的 ms/毫秒监测。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56084/56084fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="2">HPLC 条件</td>
		</tr>
		<tr>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td>列</td>
			<td>C8;2.6 µm;100Å;50 x 2.1 毫米</td>
		</tr>
		<tr>
			<td rowspan="2">守卫专栏</td>
			<td>高效液相色谱在线过滤0.5 µm;深度滤镜 x 0.004 inID</td>
		</tr>
		<tr>
			<td>过滤器 x 0.004 inID</td>
		</tr>
		<tr>
			<td>柱温:</td>
			<td>30° c</td>
		</tr>
		<tr>
			<td>移动阶段 A:</td>
			<td>0.1% FA 在水中</td>
		</tr>
		<tr>
			<td>移动阶段 B:</td>
			<td>100% ACN</td>
		</tr>
		<tr>
			<td>起始点的流动相组成</td>
			<td>60/40 (v/五)</td>
		</tr>
		<tr>
			<td>样品温度:</td>
			<td>4° c</td>
		</tr>
		<tr>
			<td>注塑量:</td>
			<td>5µL</td>
		</tr>
		<tr>
			<td>洗针</td>
			<td>80% 甲醇, 20% 水300µL</td>
		</tr>
		<tr>
			<td>流量:</td>
			<td>0.5 毫升/分</td>
		</tr>
		<tr>
			<td rowspan="7">梯度:</td>
			<td>-1 分钟: 40% B</td>
		</tr>
		<tr>
			<td>-1-2 分钟: 40%-70% B</td>
		</tr>
		<tr>
			<td>-2-2.7 分钟: 在 70% B 举行</td>
		</tr>
		<tr>
			<td>-2.7-2.8 分钟: 70%-90% B</td>
		</tr>
		<tr>
			<td>-2.8-4.0 分钟: 90% B 洗</td>
		</tr>
		<tr>
			<td>-4.0-4.1 分: 90%-40%</td>
		</tr>
		<tr>
			<td>-4.1-6.0 分钟: 举行在 40%,</td>
		</tr>
		<tr>
			<td>总运行:</td>
			<td>6分钟</td>
		</tr>
		<tr>
			<td rowspan="5">保留时间:</td>
			<td>M6 的保留时间: 1.0 分钟</td>
		</tr>
		<tr>
			<td>M1 的保留时间: 1.3 分钟</td>
		</tr>
		<tr>
			<td>滞留时间: 1.55 分钟</td>
		</tr>
		<tr>
			<td>LUMA 的保留时间: 2.3 分钟</td>
		</tr>
		<tr>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">MS 条件</td>
		</tr>
		<tr>
			<td>检测模式:</td>
			<td>电喷雾阳性</td>
		</tr>
		<tr>
			<td>离子喷雾电压:</td>
			<td>4.5 kV</td>
		</tr>
		<tr>
			<td>碰撞能量:</td>
			<td>295.9 V</td>
		</tr>
		<tr>
			<td>雾化气体 (氮气):</td>
			<td>3升/分</td>
		</tr>
		<tr>
			<td>CID 气体:</td>
			<td>230人民军</td>
		</tr>
		<tr>
			<td>干燥气体 (氮气) 流量:</td>
			<td>20升/分</td>
		</tr>
		<tr>
			<td>Q1 前杆偏置:</td>
			<td>-#1</td></tr></tbody></table>60;     5 V
		
		<tr>
			<td>ce:</td>
			<td>-25 V</td>
		</tr>
		<tr>
			<td>Q3 pre-rod 偏见:</td>
			<td>-5 V</td>
		</tr>
		<tr>
			<td>接口电流:</td>
			<td>0.1 UA</td>
		</tr>
		<tr>
			<td>去温度:</td>
			<td>250° c</td>
		</tr>
		<tr>
			<td>热块温度:</td>
			<td>400° c</td>
		</tr>
		<tr>
			<td>m/z</td>
			<td>392.49 →393</td>
		</tr>
		<tr>
			<td>M1 m/z</td>
			<td>408.49 →409</td>
		</tr>
		<tr>
			<td>M6 m/z</td>
			<td>422.47 →423</td>
		</tr>
		<tr>
			<td>LUMA m/z</td>
			<td>452.40 →453</td>
		</tr>
	

表 1: HPLC-质谱的详细情况。

Watch this Scientific Journal Video about Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cys

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Ivacaftor 和 Ivacaftor-lumacaftor 结合是两个新的 CF 药物。然而, 在他们的 PK/PD 和药理学方面仍然缺乏了解。本文提出了一种优化的 HPLC-MS 技术, 同时分析 ivacaftor 及其主要代谢产物, 以及 lumacaftor。

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8.2K 次观看.  Monash University. Ivacaftor 和 Ivacaftor-lumacaftor 结合是两个新的 CF 药物。然而, 在他们的 PK/PD 和药理学方面仍然缺乏了解。本文提出了一种优化的 HPLC-MS 技术, 同时分析 ivacaftor 及其主要代谢产物, 以及 lumacaftor。

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生物化学

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

LC-MS Analysis of Human Platelets as a Platform for Studying Mitochondrial Metabolism

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic pathways requires a model in which external factors can be well controlled, allowing for reproducible and meaningful results. Many studies employ transformed cellular models for these purposes; however, metabolic reprogramming that occurs in many cancer cell lines may introduce confounding variables. For this reason primary cells are desirable, though attaining adequate biomass for metabolic studies can be challenging. Here we show that human platelets can be utilized as a platform to carry out metabolic studies in combination with liquid chromatography-tandem mass spectrometry analysis. This approach is amenable to relative quantification and isotopic labeling to probe the activity of specific metabolic pathways. Availability of platelets from individual donors or from blood banks makes this model system applicable to clinical studies and feasible to scale up. Here we utilize isolated platelets to confirm previously identified compensatory metabolic shifts in response to the complex I inhibitor rotenone. More specifically, a decrease in glycolysis is accompanied by an increase in fatty acid oxidation to maintain acetyl-CoA levels. Our results show that platelets can be used as an easily accessible and medically relevant model to probe the effects of xenobiotics on cellular metabolism.

Here we show isolated human platelets can be used as an accessible ex vivo model to study metabolic adaptations in response to the complex I inhibitor rotenone. This approach employs isotopic tracing and relative quantification by liquid chromatography-mass spectrometry and can be applied to a variety of study designs.

人类血小板的LC-MS分析作为研究线粒体代谢的平台

Perturbed mitochondrial metabolism has received renewed interest as playing a causative role in a range of diseases. Probing alterations to metabolic ...

环境科学

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

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 ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

生物工程

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...