During preclinical drug development, a method for quantification of unlabeled compounds in blood plasma samples from treatment or pharmacokinetic studies in mice is required. In the current work, a rapid, specific, sensitive and validated liquid chromatography mass-spectrometric UHPLC-ESI-QTOF-MS method was developed for the quantification of the therapeutic compound RD2 in mouse plasma. RD2 is an all-D-enantiomeric peptide developed for the treatment of Alzheimer’s disease, a progressive neurodegenerative disease finally leading to dementia. Due to RD2’s highly hydrophilic properties, the sample preparation and the chromatographic separation and quantification were very challenging. The chromatographic separation of RD2 and its internal standard were accomplished on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm particle size) within 6.5 min at 50 °C with a flow rate of 0.5 mL/min. Mobile phases consisted of water and acetonitrile with 1% formic acid and 0.025% heptafluorobutyric acid, respectively. Ions were generated by electrospray ionization (ESI) in the positive mode and the peptide was quantified by QTOF-MS. The developed extraction method for RD2 from mouse plasma revealed complete recovery. The linearity of the calibration curve was in the range of 5.3 ng/mL to 265 ng/mL (r² > 0.999) with a lower limit of detection (LLOD) of 2.65 ng/mL and a lower limit of quantification (LLOQ) of 5.3 ng/mL. The intra-day and inter-day accuracy and precision of RD2 in plasma ranged from −0.54% to 2.21% and from 1.97% to 8.18%, respectively. Moreover, no matrix effects were observed and RD2 remained stable in extracted mouse plasma at different conditions. Using this validated bioanalytical method, plasma samples of unlabeled RD2 or placebo treated mice were analyzed. The herein developed UHPLC-ESI-QTOF-MS method is a suitable tool for the quantitative analysis of unlabeled RD2 in plasma samples of treated mice.

在临床前药物开发过程中，需要一种定量方法，该方法用于对来自小鼠治疗或药代动力学研究的血浆样品中未标记的化合物进行定量。在当前工作中，开发了一种快速，特异性，灵敏且经过验证的液相色谱质谱UHPLC-ESI-QTOF-MS方法，用于定量小鼠血浆中的治疗性化合物RD2。RD2是全D-对映体肽被开发用于治疗阿尔茨海默氏病，该疾病是最终导致痴呆的一种进行性神经退行性疾病。由于RD2具有高度亲水性，因此样品制备以及色谱分离和定量分析都非常具有挑战性。在Acquity UPLC BEH C18色谱柱（2.1×100 mm，1.7μm粒径）上，于50°C在6.5分钟内，以0.5 mL / min的流速完成RD2及其内标的色谱分离。流动相分别由水和乙腈组成，分别含1％的甲酸和0.025％的七氟丁酸。通过电喷雾电离（ESI）以正模式产生离子，并通过QTOF-MS对肽进行定量。从小鼠血浆中开发的RD2提取方法表明已完全恢复。²  > 0.999），检测下限（LLOD）为2.65 ng / mL，定量下限（LLOQ）为5.3 ng / mL。血浆中RD2的日内和日间准确度和精密度分别在-0.54％至2.21％和1.97％至8.18％之间。此外，未观察到基质效应，并且在不同条件下在提取的小鼠血浆中RD2保持稳定。使用这种经过验证的生物分析方法，对未标记的RD2或安慰剂治疗的小鼠的血浆样品进行了分析。本文开发的UHPLC-ESI-QTOF-MS方法是用于定量分析治疗小鼠血浆样品中未标记RD2的合适工具。