Hunan Creates Med Technology Co., Ltd, Changsha 410205, China.
Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, China.
J Pharm Biomed Anal. 2024 Oct 15;249:116387. doi: 10.1016/j.jpba.2024.116387. Epub 2024 Jul 28.
Baloxavir marboxil (BXM) is a cap-dependent nucleic acid endonuclease inhibitor, which exerts its antiviral effects after being metabolized to its active form baloxavir acid (BXA). Ethylenediamine tetra-acetic acid (EDTA) and heparin are the two most used anticoagulants in clinical blood sample collection to estimate drug levels in plasma. However, compared to heparin plasma, there is a lack of clinical pharmacokinetic data of BXA using EDTA anticoagulant tubes for blood collection. In the present study, an efficient, rapid, and sensitive ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for simultaneous quantification of BXM and its active metabolite BXA in human plasma with its isotopic baloxavir-d5 (BXA-d5) as internal standard (IS). Plasma samples (50 μL) were undergone using acetonitrile containing 0.1 % formic acid a precipitant. Chromatographic separation was achieved by a Waters XBridge®C8 (2.1 mm × 50 mm, 2.5 µm) column. The gradient mobile phase was 0.1 % formic acid in water (A, pH 2.8) and 0.1 % formic acid in acetonitrile (B) and delivered at a flow rate of 0.6 mL/min for 4.5 min. BXM and BXA were monitored using a positive electrospray triple quadrupole mass spectrometer (TRIPLE QUAD™ 6500+) via multiple reaction monitoring mode. The mass-to-charge ratios (m/z) were 572.2→247.0, 484.2→247.0 and 489.2→252.0 for BXM, BXA, and BXA-d5 (IS). Calibration curves exhibited excellent linearity in the range of 0.1-10 ng/mL for BXM (r > 0.996), and 0.3-300 ng/mL for BXA (r > 0.998). Within-run and between-run precisions in coefficients of variations were less than 11.62 % for BXM and less than 7.47 % for BXA, and accuracies in relative error were determined to be within -7.78 % to 5.70 % for BXM and -6.67 % to 8.56 % for BXA. Extraction recovery efficiency was 92.76 % for BXM, 95.32 % for BXA, and 99.26 % for BXA-d5, respectively. The matrix effect of BXM and BXA was in line with the requirements, where the relative deviation of the accuracy was less than 6.67 % and the precision was less than 6.69 %. The validated efficient and simple UHPLC-MS/MS method was successfully used in the pharmacokinetic study of BXM and BXA in healthy human volunteers with KEDTA and heparin tubes for blood collection. EDTA might compete with BXA for chelating metal ions and thereby decrease the plasma ratio in whole blood, leading to approximately 50 % lower measurement of pharmacokinetic parameters as compared with those obtained from heparin plasma anticoagulant tubes.
巴洛沙韦甲酯（BXM）是一种依赖于帽子的核酸内切酶抑制剂，在代谢为其活性形式巴洛沙韦酸（BXA）后发挥抗病毒作用。乙二胺四乙酸（EDTA）和肝素是临床血液样本采集中最常用的两种抗凝剂，用于估计血浆中的药物水平。然而，与肝素血浆相比，使用 EDTA 抗凝管采集血液的 BXA 的临床药代动力学数据缺乏。在本研究中，开发并验证了一种高效、快速、灵敏的超高效液相色谱-串联质谱（UHPLC-MS/MS）方法，用于同时定量人血浆中的 BXM 和其活性代谢物 BXA，以其同位素巴洛沙韦-d5（BXA-d5）作为内标（IS）。血浆样品（50 μL）用含 0.1%甲酸的乙腈沉淀。色谱分离在 Waters XBridge®C8（2.1mm×50mm，2.5μm）柱上进行。梯度流动相为 0.1%甲酸在水中（A，pH2.8）和 0.1%甲酸在乙腈中（B），流速为 0.6mL/min，持续 4.5min。BXM 和 BXA 通过正电喷雾三重四极杆质谱仪（TRIPLE QUAD™6500+）以多重反应监测模式进行监测。质荷比（m/z）分别为 572.2→247.0、484.2→247.0 和 489.2→252.0 用于 BXM、BXA 和 BXA-d5（IS）。BXM 的校准曲线在 0.1-10ng/mL 范围内表现出极好的线性（r＞0.996），BXA 的校准曲线在 0.3-300ng/mL 范围内表现出极好的线性（r＞0.998）。在 BXM 中，批内和批间精密度的变异系数小于 11.62%，BXA 的变异系数小于 7.47%，相对误差的准确度在 BXM 中为-7.78%至 5.70%，在 BXA 中为-6.67%至 8.56%。BXM 的提取回收率效率为 92.76%，BXA 的提取回收率效率为 95.32%，BXA-d5 的提取回收率效率为 99.26%。BXM 和 BXA 的基质效应符合要求，其中准确度的相对偏差小于 6.67%，精密度小于 6.69%。该验证的高效简单 UHPLC-MS/MS 方法成功用于健康志愿者使用 KEDTA 和肝素管采集血液时 BXM 和 BXA 的药代动力学研究。EDTA 可能与 BXA 竞争螯合金属离子，从而降低全血中的血浆比，导致与从肝素血浆抗凝管获得的药代动力学参数相比，测量值降低约 50%。
