Department of Pharmacy, The Affiliated LiHuiLi Hospital of Ningbo University, Ningbo, Zhejiang, China.
Laboratory Animal Centre, Wenzhou Medical University, Wenzhou, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Sep 15;1245:124272. doi: 10.1016/j.jchromb.2024.124272. Epub 2024 Aug 6.
In this experiment, a rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology was established and validated for the quantitation and pharmacokinetic analysis of eupafolin in rat plasma, utilizing licochalcone B as internal standard (IS). After liquid-liquid extraction of the analyte samples by ethyl acetate, chromatographic separation was achieved using a UPLC HSS T3 column under gradient elution conditions, with the mobile phase consisting of acetonitrile and water (with 0.1 % formic acid). Eupafolin was quantified by multiple reaction monitoring (MRM) in electrospray positive-ion mode (ESI+), employing the mass transition m/z 315.2 → 300.3 for eupafolin and m/z 285.4 → 270.3 for IS. Eupafolin demonstrated excellent linear relationship (r > 0.99) over the concentration range of 1.25-1250 ng/mL, with the lower limit of quantification (LLOQ) of the UPLC-MS/MS assay determined as 1.25 ng/mL. Method validation followed the bioanalytical method validation criteria outlined by the FDA. The accuracy of eupafolin ranged from 86.7 % to 111.2 %, and the precision was less than 12 %. The matrix effect was observed at 92.8 %-98.6 %, while the recoveries exceeded 83.2 %. The established UPLC-MS/MS assay was successfully employed for the pharmacokinetic evaluation of eupafolin in rats. The half-lives (t) were determined to be 1.4 ± 0.4 h and 2.5 ± 1.4 h for intravenous and oral administration, respectively. Notably, the bioavailability of eupafolin was relatively low (8.3 %). The optimized UPLC-MS/MS technology showed highly sensitive, selective, and effective, rendering it suitable for the pharmacokinetics of eupafolin in preclinical practice.
在这项实验中，建立并验证了一种快速灵敏的超高效液相色谱-串联质谱（UPLC-MS/MS）技术，用于定量分析和药代动力学分析大鼠血浆中的 eupafolin，内标物为 licochalcone B。经乙酸乙酯液液萃取分析物样品后，采用 UPLC HSS T3 柱在梯度洗脱条件下进行色谱分离，流动相由乙腈和水（含 0.1%甲酸）组成。采用电喷雾正离子模式（ESI+）下的多重反应监测（MRM）定量 eupafolin，其母离子-子离子跃迁质量数 m/z 315.2→300.3 用于 eupafolin，m/z 285.4→270.3 用于内标物。eupafolin 的浓度范围为 1.25-1250ng/mL 时，具有极好的线性关系（r＞0.99），UPLC-MS/MS 分析的定量下限（LLOQ）确定为 1.25ng/mL。方法验证遵循 FDA 规定的生物分析方法验证标准。eupafolin 的准确度为 86.7%-111.2%，精密度小于 12%。观察到基质效应为 92.8%-98.6%，回收率超过 83.2%。建立的 UPLC-MS/MS 分析方法成功应用于大鼠体内 eupafolin 的药代动力学评价。静脉注射和口服给药的半衰期（t）分别为 1.4±0.4h 和 2.5±1.4h。值得注意的是，eupafolin 的生物利用度相对较低（8.3%）。优化后的 UPLC-MS/MS 技术具有高灵敏度、选择性和有效性，适合于临床前实践中 eupafolin 的药代动力学研究。
