Stimultaneous determination of podophyllotoxin; quercetin and kaempferol in podophyllin by liquid chromatography Tandem Mass Spectrometry

一種定量之高效液相層析串聯式質譜法被開發，以用於分析鬼臼樹脂(Podophyllin)中成分。使用Cosmosil碳18管柱作層析分離，移動相爲0.25%甲酸－甲醇之混合液進行梯度沖提。經管柱分離後之流出液以2:3之比例，分別進入紫外光可見光檢測器及串聯質譜儀。以子代離子掃瞄鑑定鬼臼樹脂中podophyllotoxin、quercetin及kaempferol之成分，並以多重離子監測模式測定其含量。Podophyllotoxin、quercetin及kaempferol之偵測極限及定量極限分別爲2.40，8.01；2.94，9.87與3.10，10.2 ng/mL。三成分之同日間及異日間試驗相對標準偏差介於0.52-6.01%及2.48-9.88%。平均回收率各爲101.4%、98.3%及98.7%。由實驗結果可知，所開發之高效液相層析串聯式質譜法可同時偵測分析podophyllin中之podophyllotoxin、quercetin及kaempferol成分。
A quantitative high performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) was developed to analyze the constituents of podophyllin. Chromatographic separation was performed on a Cosmosil 5C18-MS (25 cm×4.6 mm I.D., 5 μm) reverse phase column using a gradient of mobile phase (0.25% formic acid-methanol). The column effluent was split 2:3 into the photodiode detector and tandem mass spectrometer. Podophyllotoxin, quercetin and kaempferol in podophyllin were identified by daughter ion scan mode and then determined their contents by multiple-reaction monitoring (MRM) mode. The limits of detection and quantitation for podophyllotoxin, quercetin and kaempferol were 2.40, 8.01; 2.94, 9.87 and 3.10, 10.2 ng/mL, respectively. The relative standard deviations of intraday and interday analyses for podophyllotoxin, quercetin and kaempferol ranged from 0.52-6.01% and 2.48-9.88%, respectively. The mean recoveries for podophyllotoxin, quercetin and kaempferol were 101.4%, 98.3% and 98.7%, respectively. The developed LC/MS/MS method was suitable for the simultaneous determinations of podophyllotoxin, quercetin and kaempferol in podophyllin.