. 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<sup cla...

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。

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			<td height="21" style="height:21px;width:480px;">IVA (Cat#S114)&#160;</td>
			<td style="width:229px;">SelleckChem (USA).&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LUMA (Cat#S1565)&#160;</td>
			<td>SelleckChem (USA).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVA-carboxylate (Cat# 510242247CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydroxymethyl-IVA (Cat# 510240849CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (MeOH, LC-MS grade),&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetonitrile (ACN, LC-MS grade)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formic acid (FA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triple-quadrupole Shimadzu 8030 LC-MS&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenomenex Kinetex (2.6 &#181;m C8 100 &#197;; 50 &#215; 2.1mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL polypropylene microcentrifuge tube (VWR).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Centrifuge 5430</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13-mm syringe filter (0.45 &#181;m nylon, GRACE, USA)&#160;</td>
			<td></td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[Phenomenex VEREX, 9 mm, PP, 300 &#181;L, PTFE/Silicone septa].&#160;</td>
			<td></td>
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		</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?...

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

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

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。

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

optimized-lc-msms-method-for-high-throughput-analysis-clinical

Research

JoVE Journal

Medicine

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。

视频: 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

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 ...

科研

医学

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

在耐药性颞叶癫痫患者卵圆孔电极记录的网络分析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

毛喉素诱导在肠组织体溶胀:一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

在非洲裔美国人中收集头发皮质醇分析样本

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

使用电子地图的光映射数据的高通量分析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

小鼠肝细胞核球状估计的高通量原位方法

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

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. ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

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