Wei Fenghuan, Singh Ravi Shankar Prasad, Fueth Matthias, Swarts Steven, Okunieff Paul, Derendorf Hartmut
Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL 32610, United States; Department of Chinese Medicine Pharmaceutics, College of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China.
Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL 32610, United States.
J Pharm Biomed Anal. 2015 Mar 25;107:82-8. doi: 10.1016/j.jpba.2014.12.025. Epub 2014 Dec 22.
Radix Phytolaccae (the dried root of Phytolacca acinosa Roxb. or Phytolacca americana L.) is widely used in East Asian countries for the treatment of inflammation-related diseases. The active component of Radix Phtolaccae is Phytolcaccagenin a triterpenoid saponin. Phytolcaccagenin has anti-inflammatory activities that exceed those of Esculentoside A and its derivatives regarding suppression of LPS-induced inflammation, and has a lower toxicity profile with less hemolysis. To date, no information is available about analytical method and pharmacokinetic studies of phytolaccagenin. To explore PK profile of this compound, a HPLC-MS/MS assay of phytolaccagenin in rat plasma was developed and validated. The method was fully validated according to FDA Guidance for industry. The detection was performed by a triple-quadrupole tandem mass spectrometer with multiple reactions monitoring (MRM) in positive ion mode via electrospray ionization. The monitored transitions were m/z 533.2>515.3 for Phytolcaccagenin, and 491.2>473.2 for I.S. The analysis was performed on a Symmetry C18 column (4.6 mm × 50 mm, 3.5 μm) using gradient elution with the mobile phase consisting of acetonitrile and 0.1% formic acid water at a flow rate of 1 ml/min with a 1:1 splitter ratio. The method was validated with a LLOQ of 20 ng/ml and an ULOQ of 1000 ng/ml. The response versus concentration data were fitted with 1/x weighting and the correlation coefficient (r) were greater than 0.999. The average matrix effect and the average extraction recovery were acceptable. This validation in rat plasma demonstrated that phytolaccagenin was stable for 30 days when stored below -20°C, for 6h at room temperature (RT, 22°C), for 12 h at RT for prepared control samples in auto-sampler vials, and during three successive freeze/thaw cycles results at -20°C. The validated method has been successfully applied to an intravenous bolus pharmacokinetic study of phytolaccagenin in male Sprague-Dawley rats (10 mg/kg, i.v.). Blood samples taken from 0 to 24h after injection were collected, and data analyzed with WinNonlin. The half-life and clearance were 1.4±0.9 h and 2.1±1.1 L/h/kg, respectively.
商陆（商陆或垂序商陆的干燥根）在东亚国家被广泛用于治疗炎症相关疾病。商陆的活性成分是商陆皂苷元，一种三萜皂苷。在抑制脂多糖诱导的炎症方面，商陆皂苷元具有超过商陆酸及其衍生物的抗炎活性，并且毒性较低，溶血作用较小。迄今为止，关于商陆皂苷元的分析方法和药代动力学研究尚无相关信息。为了探究该化合物的药代动力学特征，建立并验证了大鼠血浆中商陆皂苷元的高效液相色谱 - 串联质谱（HPLC-MS/MS）测定法。该方法根据美国食品药品监督管理局（FDA）行业指南进行了全面验证。检测通过三重四极杆串联质谱仪，采用电喷雾电离正离子模式下的多反应监测（MRM）进行。监测的跃迁对于商陆皂苷元为m/z 533.2>515.3，对于内标为491.2>473.2。分析在Symmetry C18柱（4.6 mm×50 mm，3.5μm）上进行，使用梯度洗脱，流动相由乙腈和0.1%甲酸水组成，流速为1 ml/min，分流比为1:1。该方法的定量下限（LLOQ）为20 ng/ml，定量上限（ULOQ）为1000 ng/ml。响应与浓度数据采用1/x加权拟合，相关系数（r）大于0.999。平均基质效应和平均提取回收率均可接受。在大鼠血浆中的验证表明，商陆皂苷元在-20°C以下储存30天、室温（RT，22°C）下储存6小时、自动进样瓶中制备的对照样品在室温下储存12小时以及在-20°C下进行三个连续冻融循环时均稳定。已验证的方法已成功应用于雄性Sprague-Dawley大鼠（10 mg/kg，静脉注射）中商陆皂苷元的静脉推注药代动力学研究。收集注射后0至24小时采集的血样，并用WinNonlin软件进行数据分析。半衰期和清除率分别为1.4±0.9小时和2.1±1.1 L/h/kg。
