Department of Analytical Chemistry, Ghent University , Campus Sterre, Krijgslaan 281-S12, 9000 Ghent, Belgium.
Pharmacokinetics, Dynamics & Metabolism, Janssen R&D , Turnhoutseweg 30, 2340 Beerse, Belgium.
Anal Chem. 2017 Feb 7;89(3):1907-1915. doi: 10.1021/acs.analchem.6b04388. Epub 2017 Jan 13.
Quantitative determination of the candidate drug molecule and its metabolites in biofluids and tissues is an inevitable step in the development of new pharmaceuticals. Because of the time-consuming and expensive nature of the current standard technique for quantitative metabolite profiling, i.e., radiolabeling followed by high-performance liquid chromatography (HPLC) with radiodetection, the development of alternative methodologies is of great interest. In this work, a simple, fast, sensitive, and accurate method for the quantitative metabolite profiling of an amino group containing drug (levothyroxine) and its metabolites in human plasma, based on precolumn derivatization followed by HPLC-inductively coupled plasma mass spectrometry (ICPMS), was developed and validated. To introduce a suitable "heteroelement" (defined here as an element that is detectable with ICPMS), an inexpensive and commercially available reagent, tetrabromophthalic anhydride (TBPA) was used for the derivatization of free NH-groups. The presence of a known number of I atoms in both the drug molecule and its metabolites enabled a cross-validation of the newly developed derivatization procedure and quantification based on monitoring of the introduced Br. The formation of the derivatives was quantitative, providing a 4:1 stoichiometric Br/NH ratio. The derivatives were separated via reversed-phase HPLC with gradient elution. Bromine was determined via ICPMS at a mass-to-charge ratio of 79 using H as a reaction gas to ensure interference-free detection, and iodine was determined at a mass-to-charge ratio of 127 for cross-validation purposes. The method developed shows a fit-for-purpose accuracy (recovery between 85% and 115%) and precision (repeatability <15% RSD). The limit of quantification (LoQ) for Br was approximately 100 μg/L.
定量测定生物流体和组织中的候选药物分子及其代谢物是开发新药物不可避免的步骤。由于当前定量代谢物分析的标准技术，即放射性标记后高效液相色谱（HPLC）与放射性检测，耗时且昂贵，因此开发替代方法具有重要意义。在这项工作中，开发并验证了一种基于柱前衍生化结合高效液相色谱-电感耦合等离子体质谱（ICPMS）的简单、快速、灵敏、准确的方法，用于含氨基药物（左甲状腺素）及其代谢物在人血浆中的定量代谢物分析。为引入合适的“杂元素”（定义为可通过 ICPMS 检测到的元素），使用一种廉价且市售的试剂四溴邻苯二甲酸酐（TBPA）对游离 NH 基团进行衍生化。药物分子及其代谢物中存在已知数量的 I 原子，这使得新开发的衍生化程序和基于引入 Br 的定量监测得以交叉验证。衍生物的形成是定量的，提供了 4:1 的化学计量 Br/NH 比。通过反相 HPLC 进行梯度洗脱分离衍生物。通过使用 H 作为反应气体的 ICPMS 在质荷比 79 处测定 Br，以确保无干扰检测，并在质荷比 127 处测定 I 以进行交叉验证。所开发的方法显示出适合目的的准确性（回收率在 85%至 115%之间）和精密度（重复性 <15%RSD）。Br 的定量限（LoQ）约为 100μg/L。
