Inovalab AG, Kägenstrasse 17, CH-4153 Reinach, Switzerland.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012 Feb 15;885-886:50-60. doi: 10.1016/j.jchromb.2011.12.012. Epub 2011 Dec 23.
This paper describes the development and validation of a liquid chromatography (LC)-electrospray ionization tandem mass spectrometry assay for the fully automated simultaneous determination of bosentan, a dual endothelin receptor antagonist used in the treatment of pulmonary arterial hypertension, and its three primary metabolites hydroxy bosentan (Ro 48-5033), desmethyl bosentan (Ro 47-8634), and hydroxy desmethyl bosentan (Ro 64-1056) in human dried blood spots (DBS) by use of the Sample Card And Prep (SCAP) DBS System. The system enabled the online extraction of compounds from filter paper cards without the need for punching and sample pretreatment. This was realized by automatic introduction of DBS sample cards into the LC flow via a pneumatically controlled clamp module. Using a three-column setup comprised of two pre columns for successive online DBS sample cleanup and a Synergi™ POLAR-RP C(18) analytical column for chromatographic separation under gradient conditions with a mobile phase A consisting of 1% acetic acid and a mobile phase B consisting of 1% acetic acid in methanol/2-propanol (80/20, v/v). MS/MS detection was performed in the positive multiple reaction monitoring mode using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray™ source. The total run time was 9.0min. The individual phases of online human DBS analysis were synchronized by automated valve switching. The analytical method was shown to be sensitive and selective with inter-day accuracy and precision of 91.6-108.0% and 3.4-14.6%, respectively, and it exhibited good linearity (r(2)≥0.9951 for all analytes) over the concentration range of 2ng/mL (5ng/mL for Ro 47-8634)-1500ng/mL. The analytes were stable in human DBS over 3.5 months at ambient temperature and accurate and precise results were obtained when using a blood spot volume between 20 and 30μL. Furthermore, no apparent (-8.9 to 12.6%) impact of hematocrit values ranging from 0.35 to 0.65 was observed on the quantification of the analytes. The system allowed very good recoveries of all analytes, between 83.0% and 92.3% for bosentan, between 94.4% and 100% for Ro 48-5033, between 98.0% and 100% for Ro 47-8634, and between 94.3% and 100% for Ro 64-1056. The validation demonstrated that the SCAP DBS System provides a robust automated platform for DBS analysis.
本文描述了一种液相色谱（LC）-电喷雾串联质谱法的开发和验证，用于全自动同时测定波生坦，一种用于治疗肺动脉高压的双重内皮素受体拮抗剂，及其三种主要代谢物羟波生坦（Ro 48-5033）、去甲波生坦（Ro 47-8634）和羟去甲波生坦（Ro 64-1056）在人干血斑（DBS）中的含量。该系统通过气动控制夹模块，使化合物能够在线从滤纸卡中提取，无需打孔和样品预处理。这是通过自动将 DBS 样品卡引入 LC 流中实现的。该系统采用三柱装置，包括两个预柱，用于连续在线 DBS 样品净化，以及 Synergi™ POLAR-RP C（18）分析柱，用于在梯度条件下进行色谱分离，流动相 A 为 1%乙酸，流动相 B 为 1%乙酸甲醇/2-丙醇（80/20，v/v）。采用配备 TurboIonSpray™源的 Sciex API 4000 三重四极杆 LC-MS/MS 系统，以正离子多重反应监测模式进行 MS/MS 检测。在线人 DBS 分析的各个阶段通过自动阀切换同步。该分析方法具有较高的灵敏度和选择性，日内精密度和准确度分别为 91.6-108.0%和 3.4-14.6%，在 2ng/mL（Ro 47-8634 为 5ng/mL）-1500ng/mL 的浓度范围内具有良好的线性（所有分析物的 r（2）≥0.9951）。分析物在室温下在人 DBS 中稳定 3.5 个月，当使用 20-30μL 的血斑体积时可获得准确和精确的结果。此外，在 0.35 至 0.65 的血细胞比容范围内，分析物的定量没有明显的（-8.9 至 12.6%）影响。该系统允许所有分析物的回收率非常好，波生坦为 83.0%-92.3%，Ro 48-5033 为 94.4%-100%，Ro 47-8634 为 98.0%-100%，Ro 64-1056 为 94.3%-100%。验证表明，SCAP DBS 系统为 DBS 分析提供了一个强大的自动化平台。
