Kang Nae-Won, Lee Jae-Young, Song Kwangho, Kim Min-Hwan, Yoon Soyeon, Nguyen Duy-Thuc, Kim Sungho, Kim Yeong Shik, Kim Dae-Duk
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Korea.
College of Pharmacy, Chungnam National University, Daejeon 34134, Korea.
Molecules. 2020 Apr 13;25(8):1774. doi: 10.3390/molecules25081774.
Recently, potent neuroprotective and anti-diabetic effects of 7-(3-Ethyl-cis-crotonoyloxy)-1-(2-methylbutyryloxy)-3,14-dehydro--notonipetranone (ECN), a sesquiterpenoid isolated from Linnaeus, have been elucidated. To facilitate further pre-clinical evaluation in rats, an analytical method for the determination of ECN in rat plasma was developed and optimized by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples were pretreated by the protein precipitation method with an acetonitrile solution of losartan (LST) as the internal standard. Chromatographic separation was performed using a an Octadecyl-silica (ODS) column (2.6 µm, 100 x 4.6 mm) in the isocratic mode. The mobile phase, comprising 10 mM ammonium formate in water pH 5.75) and acetonitrile (11:89, /), was eluted at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed in the multiple reaction monitoring mode with positive electrospray ionization, and the mass transitions of ECN and LST were 431.3 to 97.3 and 423.1 to 207.2, respectively. The calibration curves of spiked plasma samples were linear in the 10.0-10,000 ng/mL range ( > 0.996). The lower limit of quantification (LLOQ) was determined as 10.0 ng/mL. Validation was conducted in the LLOQ, and three quality control (QC) sample levels (10.0, 25.0, 3750, and 7500 ng/mL) were studied. Among them, the relative standard deviation for the within- and between-run precisions was under 9.90%, and the relative error of the accuracies was within the -8.13% to 0.42% range. The validated method was successfully employed to investigate the pharmacokinetic properties of ECN in rats, which revealed the linear pharmacokinetic behavior of ECN for the first time.
最近，从地锦草中分离出的一种倍半萜烯化合物7-(3-乙基-顺式巴豆酰氧基)-1-(2-甲基丁酰氧基)-3,14-脱氢-去甲异土木香酮（ECN）的强大神经保护和抗糖尿病作用已得到阐明。为便于在大鼠中进行进一步的临床前评估，采用液相色谱-串联质谱法（LC-MS/MS）建立并优化了一种测定大鼠血浆中ECN的分析方法。血浆样品采用以氯沙坦（LST）乙腈溶液为内标的蛋白沉淀法进行预处理。使用十八烷基硅烷（ODS）柱（2.6 µm，100 x 4.6 mm）在等度模式下进行色谱分离。流动相由pH 5.75的10 mM甲酸铵水溶液和乙腈（11:89，v/v）组成，以0.4 mL/min的流速洗脱。采用正电喷雾电离在多反应监测模式下进行质谱检测，ECN和LST的质量转移分别为431.3至97.3和423.1至207.2。加标血浆样品的校准曲线在10.0 - 10,000 ng/mL范围内呈线性（r > 0.996）。定量下限（LLOQ）确定为10.0 ng/mL。在LLOQ下进行验证，并研究了三个质量控制（QC）样品水平（10.0、25.0、3750和7500 ng/mL）。其中，批内和批间精密度的相对标准偏差低于9.90%，准确度的相对误差在-8.13%至0.42%范围内。该验证方法成功用于研究ECN在大鼠中的药代动力学特性，首次揭示了ECN的线性药代动力学行为。
