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><tbody><tr><td>Tacrolimus</td><td>U.S. Pharmacopeial Convention</td><td>1642802</td><td></td></tr><tr><td>D&lt;sub&gt;2&lt;/sub&gt;,&lt;sup&gt;13&lt;/sup&gt;C-Tacrolimus</td><td>Toronto Research Chemicals Inc.</td><td>F370002</td><td></td></tr><tr><td>Red blood cells</td><td>University of Colorado Hospital</td><td>W20091305500 V</td><td></td></tr><tr><td>Plasma</td><td>University of Colorado Hospital</td><td>W2017130556300Q</td><td></td></tr><tr><td>Acetone CHROMASOLV, HPLC, &amp;ge;99,9%</td><td>Sigma-Aldrich</td><td>439126-4 L</td><td></td></tr><tr><td>Acetonitrile Optima LC/MS, UHPLC-UV</td><td>Thermo Fisher Scientific</td><td>A955-4</td><td></td></tr><tr><td>Isopropanol 99.9%, HPLC</td><td>Fisher Scientific</td><td>BP2632-4</td><td></td></tr><tr><td>Methanol Optima LC/MS</td><td>Thermo Fisher Scientific</td><td>A452-4</td><td></td></tr><tr><td>Water Optima LC/MS, UHPLC-UV</td><td>Thermo Fisher Scientific</td><td>W6-4</td><td></td></tr><tr><td>Formic acid</td><td>Thermo Fisher Scientific</td><td>A118P-500</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>Zinc sulfate</td><td>Thermo Fisher Scientific</td><td>Z68-500</td><td></td></tr><tr><td>0.5 &amp;ndash; 10 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008649</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Thermo Fisher Scientific</td><td>02-682-550</td><td></td></tr><tr><td>10 &amp;ndash; 100 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008651</td><td></td></tr><tr><td>10 &amp;mu;l pipet tips with filter, sterile</td><td>Neptune</td><td>BT 10XLS3</td><td></td></tr><tr><td>100 &amp;ndash; 1,000 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008653</td><td></td></tr><tr><td>100 &amp;mu;l pipet tips with filter, sterile</td><td>Neptune</td><td>BT 100</td><td></td></tr><tr><td>1,000 &amp;mu;l pipet tips with filter, sterile</td><td>Multimax</td><td>2940</td><td></td></tr><tr><td>2 &amp;ndash; 20 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008650</td><td></td></tr><tr><td>2 ml Eppendorf tube</td><td>Thermo Fisher Scientific</td><td>02-681-258</td><td></td></tr><tr><td>20 &amp;ndash; 200 &amp;micro;l pipet, VoluMate LIQUISYSTEMS</td><td>Mettler Toledo</td><td>17008652</td><td></td></tr><tr><td>20 &amp;mu;l pipet tips with filter, sterile</td><td>GeneMate</td><td>P-1237-20</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips with filter</td><td>Multimax</td><td>2938T</td><td></td></tr><tr><td>200 &amp;mu;l pipet tips with filter, sterile</td><td>Multimax</td><td>2936J</td><td></td></tr><tr><td>50 ml Falcon tube</td><td>BD Falcon</td><td>352070</td><td></td></tr><tr><td>300 &amp;mu;l inserts for HPLC vials</td><td>Phenomenex</td><td>ARO-9973-13</td><td></td></tr><tr><td>Balance PR2002</td><td>Mettler Toledo</td><td>1117050723</td><td></td></tr><tr><td>Balances AX205 Delta Range</td><td>Mettler Toledo</td><td>1119343379</td><td></td></tr><tr><td>Bullet Blender Homogenizer</td><td>Next Advance</td><td>BBX24</td><td></td></tr><tr><td>Centrifuge Biofuge Fresco</td><td>Heraeus</td><td>290395</td><td></td></tr><tr><td>Disposable Wipes</td><td>PDI</td><td>Q55172</td><td></td></tr><tr><td>Glass v ials, 4 ml</td><td>Thermo Fisher Scientific</td><td>14-955-334</td><td></td></tr><tr><td>Glass vials, 20 ml</td><td>Thermo Fisher Scientific</td><td>B7800-20</td><td></td></tr><tr><td>Gloves, nitrile</td><td>Titan Brand Gloves</td><td>44-100S</td><td></td></tr><tr><td>HPLC vials, 9 mm, 2 ml, clear</td><td>Phenomenex</td><td>ARO- 9921-13</td><td></td></tr><tr><td>Lids for HPLC vials</td><td>Phenomenex</td><td>ARO- 8952-13-B</td><td></td></tr><tr><td>Needle, 18 G 1.5</td><td>Precision Glide</td><td>305196</td><td></td></tr><tr><td>Rack for Eppendorf tubes</td><td>Thermo Fisher Scientific</td><td>03-448-11</td><td></td></tr><tr><td>Rack for HPLC Vials</td><td>Thermo Fisher Scientific</td><td>05-541-29</td><td></td></tr><tr><td>Steel beads 0.9 &amp;ndash; 2 mm</td><td>Next Advance</td><td>SSB14B</td><td></td></tr><tr><td>Storage boxes for freezers / refrigerators</td><td>Thermo Fisher Scientific</td><td>03-395-464</td><td></td></tr><tr><td>Standard multi-tube vortexer</td><td>VWR Scientific Products</td><td>658816-115</td><td></td></tr><tr><td>Whatman Paper, 903 Protein Saver US 100/PK</td><td>GE Whatman&amp;nbsp;</td><td>2016-05</td><td></td></tr><tr><td>Autosampler</td><td>CTC PAL&amp;nbsp;</td><td>PAL.HTCABIx1</td><td></td></tr><tr><td>Binary pump, Agilent 1260 Infinity</td><td>Agilent Technologies</td><td>1260 G1312B</td><td></td></tr><tr><td>Binary pump, Agilent 1290 Infinity</td><td>Agilent Technologies</td><td>1290 G4220A</td><td></td></tr><tr><td>Micro vacuum degasser, Agilent 1260</td><td>Agilent Technologies</td><td>1260 G13798</td><td></td></tr><tr><td>Column oven,&amp;nbsp; Agilent 1290 with 2 position&amp;nbsp;</td><td>Agilent Technologies</td><td>1290 G1216C</td><td></td></tr><tr><td>Thermostated column compartment with integrated 6 port switching valve</td><td>Agilent Technologies</td><td>1290 G1316C</td><td></td></tr><tr><td>HPLC pre-column cartridge, Zorbax XDB C8 (5 &amp;micro;m particle size), 4.6 &amp;middot; 12.5 mm</td><td>Phenomenex</td><td>820950-926</td><td></td></tr><tr><td>HPLC analytical column, Zorbax Eclipse-XDB-C8 (5 &amp;micro;m particle size), 4.6 &amp;middot; 150 mm</td><td>Phenomenex</td><td>993967-906</td><td></td></tr><tr><td>Tandem Mass Spectrometer</td><td></td><td></td><td></td></tr><tr><td>API5000 MS/MS with TurboIonspray source</td><td>AB Sciex</td><td>4364257</td><td></td></tr><tr><td>Mass spectrometry software</td><td>AB Sciex</td><td>Analyst 1.5.1</td><td></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

16.8K 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

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

干血和血清斑点作为样品贮存的有用工具, 用于评估肿瘤生物标志物

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

科研

癌症研究

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

使用内核生物测定真菌孢子发生和霉菌毒素生产的定量

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

免疫与感染

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

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

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

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

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

化学

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in drug discovery studies. This can be achieved by accurately measuring the plethora of cellular interactions between a cell and drug at the single-cell level (i.e., drug uptake, metabolism, and effect). This paper describes a single-cell Raman spectroscopy and mass spectrometry (MS) platform to monitor metabolic changes of cells in response to drugs. Using this platform, metabolic changes in response to the drug can be measured by Raman spectroscopy, while the drug and its metabolite can be quantified using mass spectrometry in the same cell. The results suggest that it is possible to access information about drug uptake, metabolism, and response at a single-cell level.

This protocol presents an integrated Raman spectroscopy-mass spectrometry (MS) platform that is capable of achieving single-cell resolution. Raman spectroscopy can be used to study cellular response to drugs, while MS can be used for targeted and quantitative analysis of drug uptake and metabolism.

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

生物化学

Evaluation of Drug Sorption to PVC- and Non-PVC-based Tubes in Administration Sets Using a Pump

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters (connectors), a needle cover for protection, and other accessories. Drug sorption to tubes of administration sets is a critical issue in terms of safety and efficacy. Although drug sorption is an important factor in the quality of an administration set, there are no standard evaluation methods for the regulation of drug sorption to the tubes. Here, we describe an evaluation protocol for drug sorption to tubes of administration sets. Tubes made of polyvinyl chloride (PVC)- and non-PVC-based polymeric materials were cut to 1 m in length. Diazepam and tacrolimus were used as model drugs. In the kinetic sorption study, we selected the drug concentration and flow rate based on the clinical usage of these drugs. After the dilution of each drug in a glass bottle, the diluted drug solution was delivered through tubes of administration sets using a pump. Samples were collected in amber vials at appropriate time points and the drugs were analyzed using high-performance liquid chromatography. Drug concentrations and sorption levels to tubes of the administration sets were calculated. Acceptable criteria to ensure the quality of administration sets are recommended.

We report a method for drug sorption evaluation using a pump and model drugs (diazepam and tacrolimus). After drug analysis using high-performance liquid chromatography, drug concentrations and sorption levels in the tubes of administration sets are calculated.

药物吸附的评价PVC-和非PVC为基础的管理管设置在使用泵

Administration sets are delivery tools for the direct application of drugs into the body and are composed of a spike, a drip chamber, tubes, Luer adapters ...

医学

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

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 ...

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

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

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

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

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

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

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

生物学

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. ...