Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90596
標題: Determination of isosteroidal alkaloids in Fritillaria spp. by QuEChERS combined with Liquid Chromatography-Tandem Mass Spectrometry
QuEChERS前處理技術結合液相層析串聯質譜術於貝母中異甾體生物鹼之分析
作者: 楊宬苓
Cheng-Ling Yang
關鍵字: QuEChERS
isosteroidal alkaloids
Fritillaria
LC-MS/MS
貝母
異甾體生物鹼
液相層析串聯質譜術
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摘要: Abstract   Bulbus Fritillaria (BF), one of the derivative from the plant of various Fritillaria species, has been commonly used as expectorant and antitussive herb in traditional Chinese medicine. The value of BF is relative to its quality, but there is no reliable method to evaluate its quality. Therefore, it is necessary to develop a rapid and reliable method for classification of species of BF. In this study, the QuEChERS (quick, easy, cheap, effective, rugged, and safe) as sample preparation method and combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was proposed to determine trace alkaloids of BF. The main bioactive isosteroidal alkaloids analyzed are peimine, peiminine and peimisine. The extraction efficiency of QuEChERS were systematically studied and the optimal extraction conditions were as follow: extraction of three alkaloids by using 3 mL ethyl acetate:water=75:25 (v/v) which is containing 3% ammonium hydroxide and 1 g of ammonium carbonate then mixing by vortex condition for 5 min. The extract cleaned with 10 mg of anhydrous magnesium sulfate and 20 mg of florisil. The extracted isosteroidal alkaloids were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The quantification was performing by extracted ion chromatography and MS/MS spectra was applied for identification of isosteroidal alkaloids. The linear range for Peimisine was from 0.1 to 20 μg/g and for Peiminine and Peimine were from 0.05 to 10 μg/g with coefficients of determination (R2) above 0.9923. The limit of detection (LODs) of proposed method ranged from 0.005 to 0.03μg/g. Intra-day and inter-day precisions represented in RSD% were in range of 1.1%-17.5% and 89.6%-97.0%, respectively. The amount of Peimisine, Peiminine, Peimine in Chuan-Beimu were detected by proposed method at 4.35, 0.92, 1.39 μg/g, respectively. Principal Component Analysis (PCA) can be used to discriminate between bulbs of Beimu. The results showed the proposed method has successfully applied for determination of trace steroidal alkaloids and discriminated in the bulb of Chuan-Beimu.
川貝母為百合科(Liliaceae)貝母屬 (Fritillaria)多年生草本植物,其鱗莖可供藥用,具有止咳化痰的功效。由於川貝母為高經濟價值藥品,不肖商人會將低價格的貝母摻入其中,因此需要一快速且可信賴的方法用於川貝母的鑑定。本研究發展快速(Quick)、簡單(Easy)、便宜(Cheap)、有效 (Effective)、耐用 (Rugged) 及安全 (Safe) QuEChERS前處理技術應用於川貝母樣品中貝母辛 (Peimisine)、貝母乙素 (Peiminine) 及貝母甲素 (Pemine)等異甾體生物鹼之萃取,結合液相層析串聯質譜術分析。實驗結果QuEChERS最佳化條件於川貝母樣品為取 0.5 g 之川貝母樣品,添加 3 mL含有 3% 氨水之乙酸乙酯:水=75:25,再加入 1 g 碳酸銨,渦流震盪 5 分鐘後,以 4000 rpm 轉速離心 5 分鐘,取上清液1.8 mL,添加 20 mg 矽酸鎂及 10 mg 無水硫酸鎂,並以 14000 rpm 轉速離心 3 分鐘,取上清液1.7 mL ,並以氮氣吹乾後加入 990 μL 20% 甲醇水溶液並添加 10 μL 內標準品Solasodine,最後使用液相層析串聯質譜術以子代離子掃描模式進行偵測。於最佳化萃取條件下,偵測川貝母樣品中 Peimisine線性範圍為 0.1至20 μg/g;Peiminine 及 Peimine 的線性範圍為 0.05至10 μg/g,線性相關係數 (R2) 均在 0.9923 以上,偵測極限 (limit of detection, LOD) 在 0.005-0.03 μg/g 之間。同日內與異日間精密度之相對標準偏差介於 1.1% 至 17.5% 之間。回收率則介於 89.6% 到 97.0% 之間。本研究方法不僅可以縮短樣品前處理時間,更可減少樣品及有機溶劑之使用量,可應用於其他貝母中生物鹼檢測方法之參考。應用此開發方法於真實樣品之檢測,測得異甾體生物鹼含量 Peimisine 為 4.35 μg/g、Peiminine 為 0.92 μg/g、Peimine 為 1.39 μg/g。此外,結合主成分分析統計系統,成功應用於市售川貝母及貝母類藥材中異甾體生物鹼之分析。
URI: http://hdl.handle.net/11455/90596
文章公開時間: 2018-08-27
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