This work was supported by the United States Federal Drug Administration (FDA) contract HHSF223201310224C and the United States National Institutes of Health/FDA grant 1U01FD004573-01.

从健康人不标明身份的血液样本分别来自美国科罗拉多州大学医院（科罗拉多州Aurora）。使用去鉴定血库样本进行验证研究，以及对标准品和质量控制样品的制备被视为“豁免”是由美国科罗拉多州的多机构审查委员会（COMIRB，极光，科罗拉多）。 1.准备参考和解决方案<ol><li>购买他克莫司和内部标准的D 2，13 C-他克莫司从材料清单中所列的供应商。 <ol><li>制备储备溶液中的纯甲醇以1mg / ml的他克莫司的浓度为10微克/毫升为D 2，13 C-他克莫司的浓度。请参考材料基于三个独立的权重的股票解决方案。等分储备溶液并储存在-70℃或以下。 </li></ol></li><li>制备溶液以沉淀蛋白并提取使用甲醇/ 0.2M的硫酸锌的他克莫司在水（7：3，体积比）。这个溶液还含有内标D 2，13 C-他克莫司在2.5纳克/毫升的浓度，并用于除空白样品的提取所有样品（请参见1.3.3）的提取。 <ol><li>新鲜制备这种蛋白沉淀溶液每个萃取天并设置溶液期满在12小时。 </li></ol></li><li>校准曲线和质量控制（QC）样品的制备<ol><li>通过执行使用纯甲醇原液适当稀释制备储备溶液的他克莫司。 </li><li>以制备校准品和质量控制样品，穗20微升适当稀释的原液进入EDTA全血，孵育在37℃下轻轻摇动的水浴中，以允许均匀分布的他克莫司成血细胞成分20分钟，等分到1.5毫升息肉与锥底ropylene管和单元的盖子。确保有机溶剂的相对量不超过5％。 </li><li>点50微升加标全血的插入用吸管滤波器卡每一圆圈的中间。 </li><li>干燥血斑点滤波器卡在RT搅拌3小时。 </li><li>制备在人EDTA全血的他克莫司校准标准以1，2.5，5，10，25，和50ng / ml的他克莫司的浓度。准备提取样校准标准，包含内标D 2，13 C-他克莫司（“零样本”）的蛋白质沉淀溶液空白样品。 </li><li>制备QC样品中人类EDTA全血，在0，2，4，20，40纳克/毫升的浓度。准备一个空白样品。在对比的是与含有内标D 2沉淀中提取的QC样品，13 C-他克莫司，提取液用蛋白沉淀溶液，这是否空白样品不包含内标D 2，13 C-他克莫司（“空白样品”）。 </li></ol></li><li>采集临床样品<ol><li>在43,44描述收集干血斑。 </li></ol></li></ol> 2.他克莫司的提取干血斑样本<ol><li>目视检查干燥血点，以确保可接受的样品的质量和体积45。 </li><li>冲压中心过滤卡用6毫米的打孔机上的血点。 注意：冲头的质量可能通过称重来监测。所涉及的冲压饱和滤片的重量平均5.02毫克±0.09毫克（范围：4.83- 5.14毫克，N = 12）。 </li><li>将光盘插入1.5毫升聚丙烯管与锥形底和管理单元的盖子。 </li><li>添加20〜30发子弹，每管。 </li><li>加入500微升蛋白沉淀溶液（甲醇：0.2M的硫酸锌，7：3，体积：体积，用2.5毫微克/毫升内标）到每个管中。对于抽空白样品的作用，可使用蛋白质沉淀溶液而无需内部标准。 </li><li>均质光盘中的子弹搅拌机1分（最高速度，设置为“10”）。 </li><li>摇晃样品在RT上多管涡旋（最大速度，设置为“10”）10分钟。 </li><li>离心样品在16000×g离心4℃10分钟。 </li><li>转移上清液到装有300微升插入玻璃HPLC小瓶。使用预切口特氟隆密封。 注意：提取样品可以贮存于-20℃或以下直到LC-MS / MS分析。 </li></ol> 3，LC-MS / MS分析<ol><li>负载100微升所提取样品的上清液到C8盒萃取柱，并用一个7：在水3比为0.1％甲酸：乙腈以5ml /分钟的流量为1分钟。切换阀的连接示于图2和梯度由提取泵表 1运行</st荣&gt;。 </li><li>此后，激活导致从预柱的分析物的反冲洗到分析柱切换阀。 </li><li>设置柱温箱到65℃。 </li><li>洗脱来自分析柱使用表1中所示的流速和梯度的分析物。 </li><li> 经由涡轮电喷雾离子源连接分析柱到一个串联质谱仪。 根据表2调整质谱仪的关键参数。 </li><li>检测正离子（[M +的Na] +）的多反应模式（MRM）。使用下面的离子转换为量化：他克莫司：M / Z（质量/电荷）= 826.6→616.2和D 2，13 C-他克莫司：M / Z = 829.6→619.2。 注：总运行时间为4.6分钟。 </li></ol> 4.定量<ol><li>对于每次运行，基于在1.3.5和制备的校准器产生标准曲线包括在每一个分析运行。 <ol><li>生成绘制名义浓度与分析物的响应因子（峰面积[分析物] /峰面积[内标]）使用质谱仪软件校准曲线。 </li><li>使用二次拟合结合1 / X加权拟合校准。 </li></ol></li><li>要量他克莫司在干血斑融入提取的MRM色谱图的他克莫司和内标峰。计算响应因子的他克莫司（峰面积[分析物] /峰面积[内标]），并利​​用质谱分析软件的标准曲线进行比较。 </li></ol> 5.验证程序<ol><li>检测（LLOD）下限和定量（LLOQ）下限。 <ol><li>考虑的最低他克莫司浓度的峰值 - 噪声比为4：作为检测（LLOD）的下限1。定义量化（LLOQ）的下限为与精确度等于或小于±20％的偏差更好从标称浓度和精度等于或优于20％（变异系数）校正曲线的最低浓度。 </li></ol></li><li>区域内和日间准确度和精密度。 <ol><li>测试的准确度和精确度以2纳克/毫升（QC1），4纳克/毫升（QC2），20纳克/毫升（QC3）和40纳克/毫升（QC4）4的浓度水平。 </li><li>准备在每个验证一天质控样品中人类EDTA全血，干的过滤器卡，提取，并按上述方法进行分析。 </li><li>确定日内准确度和精密度，每个QC浓度水平6个样品。 </li><li>评估日间精密度和准确度超过20天。测量每个QC水平每天4个样品。 </li><li>对每一天的质控样品分析两个校准曲线在一起。 </li><li>计算日内准确性标称浓度％（每浓度水平六个样本，请参见5.2.2节）。计算器乌拉特精度的方差（CV％）的系数。 </li><li>考虑日内精度上可接受的，如果它落入接受限制的85％至115％的标称浓度。考虑日内精密度接受的，如果它等于或优于15％的CV（变异系数）。 </li><li>计算日间准确度和精密度的平均值在20天的验证分析每个QC浓度水平。 </li><li>考虑平均日间精度上可接受的，如果它落入接受限制的85％至115％的标称浓度。考虑日间精密度接受的，如果它等于或优于15％的CV（变异系数）。 </li></ol></li><li>排除基质干扰。 <ol><li>对于可能由矩阵信号造成干扰的排除，分析空白干血斑（8种不同的个人，最好是4男4女）。 </li><li>目测离子色谱图。如果保留时间内的峰检测他克莫司的窗口，整合并与空白试样中他克莫司峰的曲线下比较各自领域掺入他克莫司在LLOQ。空白样品中的峰的面积是不应该超过这些他克莫司在LLOQ的15％。 </li></ol></li><li>离子抑制/离子增强。 <ol><li>使用柱后输注方案所描述45，以评估所造成的共洗脱基质组分离子抑制/离子增强的潜在干扰。 </li><li>注入他克莫司以溶解在0.1％甲酸10微克/ ml的浓度：甲醇（30:70，体积/体积）柱后，以10微升/分的速度。 <ol><li>连接通过分析柱和质谱仪的电源之间T形管的注射器泵。 </li><li>监测MRM离子的MS / MS信号强度为他克莫司和它的内部标准（M / Z = 826.6→616.2和m / z = 829.6→619.2）注射为Extrac的后泰德空白样品（n =来自不同个体的8个样本）。 注：在没有离子抑制/离子增强引起输注分析物的连续信号不应该受注射空白基质，同时离子抑制使信号和离子增强的峰值的倾角。 </li></ol></li></ol></li><li>结转。 <ol><li>通过分析提取的最高校准后的空白样品评估潜在的结转（50纳克/毫升，N = 6）。 </li><li>目测离子色谱图。如果他克莫司的保留时间窗内的峰被检测到，集成和与那些空白样品在他克莫司的峰的曲线下比较它们的区域掺入他克莫司在LLOQ。空白样品中的峰的面积是不应该超过这些他克莫司在LLOQ的15％。 </li></ol></li><li>提取回收率。 <ol><li>通过提取QC SAM后比较分析的信号来确定回收率普莱斯在所有四个浓度（每组n = 6浓度）与那些空白干血斑掺有提取后相应的金额的他克莫司。 </li><li>准备四组的QC（浓度水平：2，4，20日，40纳克/毫升）。 </li><li>准备另4套相应的“回收试验样品”通过发现50微升的空白EDTA全血到滤纸卡和干燥2小时的。 </li><li>此后来说，无论是质量控制和空白“回收试验样品”，切出整个血斑过滤卡上用剪刀将得到的光盘插入聚丙烯管，锥底和管理单元的盖子。 </li><li>抽取的所有样品。 </li><li>转移上清液（400微升）到玻璃HPLC小瓶。 </li><li>他克莫司添加原液到空白“提取的恢复试验样品”，达到2,4，20和40纳克/毫升（4微升200，400，2000，4000纳克/毫升的他克莫司的库存溶胶浓度utions 400微升上清液）。 </li><li>后的LC-MS / MS分析，二者QC样品和“恢复试验样品”相应的浓度在比较的信号（恢复（％）=信号样品添加后萃取×100之前提取/信号样品添加）。 </li></ol></li><li>稀释的完整性。 <ol><li>使用建立稀释完整性样品掺入分析物，在500，250和100纳克/毫升。 </li><li>提取后，使用稀释的蛋白沉淀溶液样品（1:10，每组n =浓度级别3）。 </li><li>计算从标称浓度的偏差。考虑落入85％的名义接受-115％的结果。 </li></ol></li><li>稳定性。 <ol><li>调查使用QC样品在所有四个浓度（n = 4时每个浓度）在不同时间点和不同的存储条件下分析稳定性。 </li><li>比较存储与标称值后的结果。考虑řesults落在85％的名义接受-115％的。 </li><li>建立样品稳定性1周在环境温度下，1周，在4℃，1个月-20℃1个月-80℃。 </li><li>过三个周期测试冻融稳定性（-20℃）。测试样品中提取和自动进样器的稳定性通过将样品放入恒温自动进样器调节到4℃。 72小时后注射样品。 </li></ol></li></ol>

The authors have nothing to disclose.

虽然，如上所述，基于干血斑的治疗药物和他克莫司的粘附监测的概念是有吸引力的，也有超出那些通常与他克莫司的静脉EDTA全血样品中的LC-MS / MS分析相关联的分析挑战。这些包括，但不限于一个事实，即基质是毛细管全血浸透到这里使用的过滤卡材料和低血容量（20微升）的棉短绒材料。尽管如此，在中心实验室高通量分析需要快速和可靠的提取方法，其导致缺乏基质干扰和基质效应结合一个...

他克莫司是一种强效immonosuppressant 1-7具有一个大环内酯结构8（ 图1）。由于顺 -该CN键的反异构它形成在溶液9两种旋转异构体，可通过反相高效液相色谱法进行分离（HPLC），他克莫司是亲脂性和可溶于醇（甲醇653克/升，乙醇：355克/升），卤代烃（氯仿：573克/升）和乙醚。它是难溶于脂族烃（己烷：0.1克/升和水（pH 3：0.0047克/升）9所述的分子不包含任何发色团并且其紫外吸收最大值是通过抑制神经钙的192纳米的他克莫司的行为。其作用的机制已经在参考文献10,11综述。它是目前使用在80％以上的固体器官移植患者的在美国12。 他克莫司的治疗指数是considered可窄13。此外，他克莫司的剂量和血液浓度之间的相关性差和药代动力学是可变14,15。治疗药物监测指导剂量的他克莫司在移植患者因此一般的临床实践16-20。的目标是保持他克莫司血液中的浓度的预定义治疗范围内。下面的治疗范围他克莫司血液中的浓度可导致慢性或急性同种异体免疫反应的活性增加，而浓度大于治疗窗口增加用于过免疫抑制，癌症和毒性，如肾毒性，神经毒性，高血压和糖尿病的风险。他克莫司的药代动力学高个体内的变化可能是有害的既移植器官和患者存活21,22。而他克莫司的药代动力学的个体间变异主要是由CYP3A5基因多态性，原因个体内变异包括但不限于，药物与药物，疾病-药物和食品药物相互作用14,15。也缺乏坚持免疫抑制治疗药物治疗是一个促进因素，一个重要原因移植损失23,24。 这些考虑表明，频繁家治疗药物和他克莫司的全血浓度的粘附监测可能是有益的，以确保患者在任何时候所需治疗窗口内的他克莫司的曝光。但是，物流和更频繁的治疗药物监测的成本，因为它是目前的临床实践15是令人望而却步。其中一个原因是，该患者具有看到一个抽血有绘制所需的静脉血样。干血斑最近成为一个有吸引力的概念25-28。经过简单的手指贴在病人聚集在一个特殊的过滤纸卡，后血斑有d血滴里德，它可以邮寄到中心实验室用于他克莫司的分析和任何其他的免疫抑制剂，该患者可能目前服用。这已经成为可能的，因为高度敏感和特异的LC-MS / MS测定法他克莫司和其他免疫抑制剂的定量在非常小的血液量的发展，如干燥血点（通常是20微升血液）25,29-43。另一个优点是，微创，低体积的样品收集策略诸如干血斑大大方便治疗药物的监测和药代动力学研究中的儿童小28。 他克莫司通常以静脉EDTA全血15。原因是他克莫司广泛分布成血细胞和临床研究报道他克莫司谷浓度在血液之间有更好的相关性比血浆的临床事件15,18。相比较而言，Ta的分析crolimus在干燥血点是基于毛细血与该滤纸基质混合。此呈现在他克莫司和潜在的干扰与LC-MS / MS分析的溶解方面的挑战。这里，我们提出基于使用结合的子弹搅拌器具有高流动性的在线列取样清理过程和LC-MS / MS分析的干燥血点的均质建立和验证实验。到今天为止，此法已成功地用于五千余他克莫司干血斑样本的定量分析在临床试验中坚持监测。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Reference Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tacrolimus</td>
			<td>U.S. Pharmacopeial Convention</td>
			<td>1642802</td>
		</tr>
		<tr>
			<td>D2,13C-Tacrolimus</td>
			<td>Toronto Research Chemicals Inc.</td>
			<td>F370002</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Test Materials</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cells</td>
			<td>University of Colorado Hospital</td>
			<td>W20091305500 V</td>
		</tr>
		<tr>
			<td>Plasma</td>
			<td>University of Colorado Hospital</td>
			<td>W2017130556300Q</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solvents</td>
			<td></td>
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		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone CHROMASOLV, HPLC, &#8805; 99,9 %</td>
			<td>Sigma-Aldrich</td>
			<td>439126-4 L</td>
		</tr>
		<tr>
			<td>Acetonitrile Optima LC/ MS, UHPLC- UV</td>
			<td>Thermo Fisher Scientific</td>
			<td>A955-4</td>
		</tr>
		<tr>
			<td>Isopropanol 99.9 %, HPLC</td>
			<td>Fisher Scientific</td>
			<td>BP2632-4</td>
		</tr>
		<tr>
			<td>Methanol Optima LC/ MS</td>
			<td>Thermo Fisher Scientific</td>
			<td>A452-4</td>
		</tr>
		<tr>
			<td>Water Optima LC/ MS, UHPLC- UV</td>
			<td>Thermo Fisher Scientific</td>
			<td>W6-4</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other Chemicals</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>A118P-500</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
		</tr>
		<tr>
			<td>Zinc sulfate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Z68-500</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Instruments and Consumables</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 &#8211; 10 &#181;l pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008649</td>
		</tr>
		<tr>
			<td>1,5 mL- Eppendorf tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-682-550</td>
		</tr>
		<tr>
			<td>10 &#8211; 100 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008651</td>
		</tr>
		<tr>
			<td>10 &#956;L- pipet tips with filter, sterile</td>
			<td>Neptune</td>
			<td>BT 10XLS3</td>
		</tr>
		<tr>
			<td>100 &#8211; 1000 &#181;l pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008653</td>
		</tr>
		<tr>
			<td>100 &#956;L- pipet tips with filter, sterile</td>
			<td>Neptune</td>
			<td>BT 100</td>
		</tr>
		<tr>
			<td>1000 &#956;L- pipet tips with filter, sterile</td>
			<td>Multimax</td>
			<td>2940</td>
		</tr>
		<tr>
			<td>2 &#8211; 20 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008650</td>
		</tr>
		<tr>
			<td>2 mL- Eppendorf tube</td>
			<td>Thermo Fisher Scientific</td>
			<td>02-681-258</td>
		</tr>
		<tr>
			<td>20 &#8211; 200 &#181;L pipet, VoluMate LIQUISYSTEMS</td>
			<td>Mettler Toledo</td>
			<td>17008652</td>
		</tr>
		<tr>
			<td>20 &#956;L- pipet tips with filter, sterile</td>
			<td>GeneMate</td>
			<td>P-1237-20</td>
		</tr>
		<tr>
			<td>200 &#956;L- pipet tips with filter</td>
			<td>Multimax</td>
			<td>2938T</td>
		</tr>
		<tr>
			<td>200 &#956;L- pipet tips with filter, sterile</td>
			<td>Multimax</td>
			<td>2936J</td>
		</tr>
		<tr>
			<td>50 mL- Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
		</tr>
		<tr>
			<td>300 &#956;L inserts for&#160; HPLC vials</td>
			<td>Phenomenex</td>
			<td>ARO-9973-13</td>
		</tr>
		<tr>
			<td>Balance PR2002</td>
			<td>Mettler Toledo</td>
			<td>1117050723</td>
		</tr>
		<tr>
			<td>Balances AX205 Delta Range</td>
			<td>Mettler Toledo</td>
			<td>1119343379</td>
		</tr>
		<tr>
			<td>Bullet Blender Homogenizer</td>
			<td>Next Advance</td>
			<td>BBX24</td>
		</tr>
		<tr>
			<td>Centrifuge Biofuge Fresco</td>
			<td>Heraeus</td>
			<td>290395</td>
		</tr>
		<tr>
			<td>Disposable Wipes</td>
			<td>PDI</td>
			<td>Q55172</td>
		</tr>
		<tr>
			<td>Glass v ials, 4 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-955-334</td>
		</tr>
		<tr>
			<td>Glass vials, 20 mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>B7800-20</td>
		</tr>
		<tr>
			<td>Gloves, nitrile</td>
			<td>Titan Brand Gloves</td>
			<td>44-100S</td>
		</tr>
		<tr>
			<td>HPLC vials, 9 mm, 2 mL, clear</td>
			<td>Phenomenex</td>
			<td>ARO- 9921-13</td>
		</tr>
		<tr>
			<td>Lids for HPLC vials</td>
			<td>Phenomenex</td>
			<td>ARO- 8952-13-B</td>
		</tr>
		<tr>
			<td>Needle, 18G 1.5</td>
			<td>Precision Glide</td>
			<td>305196</td>
		</tr>
		<tr>
			<td>Rack for Eppendorf tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-448-11</td>
		</tr>
		<tr>
			<td>Rack for HPLC Vials</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-541-29</td>
		</tr>
		<tr>
			<td>Steel beads 0.9 &#8211; 2 mm</td>
			<td>Next Advance</td>
			<td>SSB14B</td>
		</tr>
		<tr>
			<td>Storage boxes for freezers / refrigerators</td>
			<td>Thermo Fisher Scientific</td>
			<td>03-395-464</td>
		</tr>
		<tr>
			<td>Standard multi-tube vortexer</td>
			<td>VWR Scientific Products</td>
			<td>658816-115</td>
		</tr>
		<tr>
			<td>Whatman Paper, 903 Protein Saver US 100/PK</td>
			<td>GE Whatman&#160;</td>
			<td>2016-05</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC Equipment and Columns</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Autosampler</td>
			<td>CTC PAL&#160;</td>
			<td>PAL.HTCABIx1</td>
		</tr>
		<tr>
			<td>Binary pump, Agilent 1260 Infinity</td>
			<td>Agilent Technologies</td>
			<td>1260 G1312B</td>
		</tr>
		<tr>
			<td>Binary pump, Agilent 1290 Infinity</td>
			<td>Agilent Technologies</td>
			<td>1290 G4220A</td>
		</tr>
		<tr>
			<td>Micro vacuum degasser, Agilent 1260</td>
			<td>Agilent Technologies</td>
			<td>1260 G13798</td>
		</tr>
		<tr>
			<td>Column oven,&#160; Agilent 1290 with 2 position&#160;</td>
			<td>Agilent Technologies</td>
			<td>1290 G1216C</td>
		</tr>
		<tr>
			<td>Thermostated column compartment with integrated 6 port switching valve</td>
			<td>Agilent Technologies</td>
			<td>1290 G1316C</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC pre-column cartridge, Zorbax XDB C8 (5 &#181;m particle size), 4.6 &#183; 12.5 mm</td>
			<td>Phenomenex</td>
			<td>820950-926</td>
		</tr>
		<tr>
			<td>HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 &#181;m particle size), 4.6 &#183; 150 mm</td>
			<td>Phenomenex</td>
			<td>993967-906</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tandem Mass Spectrometer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>API5000 MS/MS with TurboIonspray source</td>
			<td>AB Sciex</td>
			<td>4364257</td>
		</tr>
		<tr>
			<td>Mass spectrometry software</td>
			<td>AB Sciex</td>
			<td>Analyst 1.5.1</td>
		</tr>
	</tbody>
</table>

quantification of the immunosuppressant tacrolimus on dried blood spots using lc-ms/ms

钙调神经磷酸酶抑制剂他克莫司是在美国实体器官移植后的免疫抑制大多数治疗方案的基石。他克莫司是一个窄的治疗指数的药物，因此需要治疗药物的监测，并根据它的全血谷浓度调整剂量。为方便家庭治疗药物，并坚持监测，干血斑的集合是一个有吸引力的概念。手指棒后，病人收集在滤纸上血滴在家里。血后进行干燥，则邮寄到分析实验室，其中他克莫司是用高效液相色谱法 - 串联质谱法量化（HPLC-MS / MS）在一个简单的手动蛋白质沉淀步骤和在线柱萃取组合。 他克莫司分析，一个6毫米光盘从血液点的饱和点刻。血斑用一颗子弹搅拌机均质第二然后蛋白沉淀，用甲醇/ 0.2M的硫酸锌含有内标D 2，13 C-他克莫司。涡旋和离心后，将100μl上清液注入到一个在线萃取柱中，用5毫升/分钟0.1甲酸/乙腈（7:3，体积比），持续1分钟。此后，切换阀被激活，分析物被反冲洗到分析柱（并用0.1％甲酸/乙腈梯度分离）。他克莫司被量化中使用的串联质谱仪的阳性反应的多模式（MRM）。 该测定是从1至50ng / ml的线性。批间变异性（3.6％-6.1％）和准确度（91.7％-101.6％）的评估20天以上达到验收标准。平均提取回收率为95.5％。有没有相关的结转，基质干扰和基体效应。他克莫司是稳定的干血斑在RT和在4℃下1周。在提取样本自动进样器是稳定的，在4℃下至少72小时。

空白样品的代表离子色谱，样品掺入在量化和患者样品的下限示于图3。 校准曲线检测的下限为0.5 ng / ml和定量的下限为1.0毫微克/毫升。把15ng / ml的被选为最高校准器更高的浓度不太可能在临床上正常的情况下达成。 校准曲线在每个验证天在人EDTA全血新鲜制备的，在过滤器上卡干燥和用萃取甲醇/ 0.2M的硫酸锌（70...

Watch this Scientific Journal Video about Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS at JoVE.com

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

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 ...

quantification-immunosuppressant-tacrolimus-on-dried-blood-spots

Research

JoVE Journal

Medicine

16.7K 次观看.  University of Colorado, Anschutz Medical Campus. 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.

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

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

科研

医学

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Dried Blood Spot Collection of Health Biomarkers to Maximize Participation in Population Studies

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spots meet this need, and can be collected in the field with high response rates. These elements are particularly important in longitudinal study designs including interventions where attrition is critical to avoid, and high response rates improve the interpretation of results. Dried Blood Spot sample collection is simple, quick, relatively painless, less invasive then venipuncture, and requires minimal field storage requirements (i.e.&#160;samples do not need to be immediately frozen and can be stored for a long period of time in a stable freezer environment before assay). The samples can be analyzed for a variety of different analytes, including cholesterol, C-reactive protein, glycosylated hemoglobin, numerous cytokines, and other analytes, as well as provide genetic material. DBS collection is depicted as employed in several recent studies.

Biomarkers are directly-measured biological indicators of disease or health. In population and social sciences, biomarkers need to be easy to obtain, transport, and analyze. Dried Blood Spot (DBS) collection meets this need, can be collected in the field with high response rates&#160;and analyzed for a variety of biomarkers.

干生生物标志物的血斑收藏最大限度地参与人口研究

Biomarkers are directly-measured biological indicators of disease, health, exposures, or other biological information. In population and social sciences, ...

Absolute Quantification of A&#946;1-42 in CSF Using a Mass Spectrometric Reference Measurement Procedure

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the diagnosis of AD is based on cognitive tests and mental state exams, but the peptide amyloid-beta (A&#946;) in cerebrospinal fluid (CSF) is increasingly used in clinical trials and settings. As for most protein and peptide biomarkers, quantification is performed using antibody-based techniques, such as enzyme-linked immunosorbent assay (ELISA). However, intra- and inter-laboratory variability in these assays hamper its use as a diagnostic marker in clinical routine.
An antibody-independent Reference Measurement Procedure (RMP) was developed based on solid-phase extraction (SPE) and liquid chromatography (LC)-tandem mass spectrometry (MS/MS), where stable, isotope-labeled A&#946; peptides were used as internal standards, enabling absolute quantification. A high-resolution quadrupole-orbitrap hybrid instrument was used for the measurements. The method allows for the quantification of CSF A&#946;1-42 between 150-4,000 pg/mL.

Absolute Quantification of A&amp;#946;&lt;sub&gt;1-42&lt;/sub&gt; in CSF Using a Mass Spectrometric Reference Measurement Procedure

A reference measurement procedure for the absolute quantification of A&#946;1-42 in human CSF based on solid-phase extraction and liquid chromatography tandem mass spectrometry is described.

Aβ的绝对定量<sub&gt; 1-42</sub&gt;在CSF使用质谱参考测量程序

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly and accounts for 60-80% of all cases of dementia. Currently, the ...

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

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and 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 ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

应用 Ameroid 的猪慢性心肌缺血模型

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

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. ...

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation

Magnetic resonance imaging (MRI) is an important tool for the clinical assessment of cardiovascular morphology and heart function. It is also the recognized standard-of-care for blood flow quantification based on phase contrast MRI. While such measurement of blood flow has been possible in adults for decades, methods to extend this capability to fetal blood flow have only recently been developed.
Fetal blood flow quantification in major vessels is important for monitoring fetal pathologies such as congenital heart disease (CHD) and fetal growth restriction (FGR). CHD causes alterations in the cardiac structure and vasculature that change the course of blood in the fetus. In FGR, the path of blood flow is altered through the dilation of shunts such that the oxygenated blood supply to the brain is increased. Blood flow quantification enables assessment of the severity of the fetal pathology, which in turn allows for suitable&#160;in utero&#160;patient management and planning for postnatal care.
The primary challenges of applying phase contrast MRI to the human fetus include small blood vessel size, high fetal heart rate, potential MRI data corruption due to maternal respiration, unpredictable fetal movements, and lack of conventional cardiac gating methods to synchronize data acquisition. Here, we describe recent technical developments from our lab that have enabled the quantification of fetal blood flow using phase contrast MRI, including advances in accelerated imaging, motion compensation, and cardiac gating.

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.