In this study, a simple and sensitive analytical method based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated for the determination of neferine in rat plasma. After acetonitrile-mediated protein precipitation, the samples were separated on an Acquity BEH C₁₈ column (2.1 × 50 mm, 1.7 μm) maintained at 40°C. The mobile phase comprising 0.1% formic acid in water and acetonitrile was delivered at a flow rate of 0.4 ml/min. The mass detection was conducted using multiple reaction monitoring mode with ion transitions at 625.4 > 206.3 and m/z 622.9 > 380.9 for neferine and internal standard, respectively. The assay was demonstrated to be linear over the concentration range of 0.5–1,000 ng/ml, with correlation coefficient >0.999 (r > 0.999). The validated method was further applied to the pharmacokinetic study of neferine in rat plasma. In addition, the metabolism of neferine was investigated using high-resolution mass spectrometry. A total of six metabolites from rat liver microsomes and plasma were detected and their structures were identified according to their fragment ions. The proposed metabolic pathways of neferine were demethylation, dealkylation, dehydrogenation and glucuronidation.

中文翻译：

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通过 LC-MS/MS 和 LC-HRMS 测定大鼠中奈芬碱的药代动力学、生物利用度和代谢

在本研究中，开发并验证了一种基于液相色谱-串联质谱 (LC-MS/MS) 的简单灵敏的分析方法，用于测定大鼠血浆中的奈芬碱。在乙腈介导的蛋白质沉淀后，样品在保持在 40°C 的 Acquity BEH C ₁₈柱 (2.1 × 50 mm, 1.7 μm) 上分离。包含在水中的 0.1% 甲酸和乙腈的流动相以 0.4 ml/min 的流速输送。使用多反应监测模式进行质量检测，其中 neferine 和内标的离子跃迁分别为 625.4 > 206.3 和m/z 622.9 > 380.9。该测定被证明在 0.5–1,000 ng/ml 的浓度范围内呈线性，相关系数 >0.999 ( r > 0.999)。经验证的方法进一步应用于大鼠血浆中奈芬碱的药代动力学研究。此外，使用高分辨率质谱法研究了 neferine 的代谢。共检测到来自大鼠肝微粒体和血浆的六种代谢物，并根据其碎片离子鉴定了它们的结构。提出的neferine代谢途径是去甲基化、脱烷基化、脱氢和葡萄糖醛酸化。