以基質輔助雷射脫附游離飛行時間質譜法開發一個具有大通量且快速篩選脫氧雪腐鐮刀菌烯醇的偵測技術

The Development of High Throughput Screening Technique for Deoxynivalenol by Matrix-Assisted Laser Desorption/Inoization Time-of -Flight Mass Spectrometry

林美文(Mei-wun Lin)

指導教授 ： 陳政男

朝陽科技大學/理工學院/應用化學系/碩士(2009年)

摘要

脫氧雪腐鐮刀菌烯醇（deoxynivalenol）也稱為DON或嘔吐毒素（vomitoxin or Vomiting toxin，vT），是自然產生於玉米、小麥和大麥裡的新月毒素（trichothecenes）之中最常見且豐富的毒素。傳統的分析方法在樣品製備及偵測過程是成本高昂且相當耗費時間，因此並不適合做高通量的篩選。到目前為止，台灣官方分析脫氧雪腐鐮刀菌烯醇的方法，是使用免疫親和管柱結合高效能液相層析法（HPLC），其偵測極限 60 ppb；若要高通量的檢測樣品，其分析時間費時且免疫親和管柱的成本較高，如果有一個低成本且具有高通量的篩選方法，則可篩選掉大部分未污染的樣品，節省不必要的高昂定量分析所需金錢與時間。
在這篇論文中，我們發表一個以基質輔助雷射脫附游離飛行時間質譜法（MALDI-TOF MS）為主的快速且高通量篩選DON的分析方法。首先，利用矽藻土為主的樣品製備方法既簡單且成本低廉，而MALDI-TOF MS本身具有高通量的特性。使用的離子液體基質Et3N-a-CHCA在低質量範圍時可降低背景雜訊的干擾，並有良好的均勻性；且加入氯化鉀（KCl）鹽類後，由於鉀離子（K+ cationization）關係使脫氧雪腐鐮刀菌烯醇的訊號增強。使用此方法的樣品製備成本只要台幣10元左右，平均分析一個樣品也只要1分鐘的時間，偵測極限可低至10 ppb。

關鍵字：脫氧雪腐鐮刀菌烯醇，基質輔助雷射脫附游離飛行時間質譜法。

關鍵字

脫氧雪腐鐮刀菌烯醇 ； 基質輔助雷射脫附游離飛行時間質譜法

並列摘要

Deoxynivalenol (DON or vomitoxin) is a mycotoxin and is one of the most common and abundant naturally occurring trichothecenes found in corn, wheat, and barley. Conventional analysis methods in the sample preparation and detection process is cost and time consuming, and therefore not suitable for high throughput screening. In Taiwan, the official analysis method for Deoxynivalenol is using immunoaffinity column coupling with high performance liquid chromatography (HPLC) which has the detection limit of 60 ppb. The cost of the specific column and the time consuming are not good for high throughput analysis. If there is a low cost and high throughput screening methods to filter out most uncontaminated samples, a lot of time consuming and cost would be avoided.
Here we report a low cost and high throughput screening method for the analysis of Deoxynivalenol using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). First, This method includes two part, sample preparation method based on celite is simple and low cost. Second, high throughput of the MALDI-TOF MS supply a detection. An ionic liquid matrix (Et3N-a-CHCA) is used to obtain low matrix noise mass spectra for low molecular weight Deoxynivalenol and the Deoxynivalenol signal was enhanced by adding KCl via K+ cationization. The method of sample preparation cost for each sample is NT$ 10.0 dollars, and a sample analysis time is only 1 min, detection limit can reach to 10 ppb.

Key words：deoxynivalenol, MALDI-TOF MS

並列關鍵字

deoxynivalenol ； MALDI-TOF MS

參考文獻

1. Petra Blechova, Pavla Havlova, Dagmar Gajdosova, Josef Havel1, “New Possibilities of Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry to Analyze Barley Malt Quality. Highly Sensitive Detection of Mycotoxins,” Environmental Toxicology., pp. 403-408 (2006).

2. Berthiller, F., Schuhmacher, R., Buttinger, G., Krska, R., “Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry,” J Chromatogr A., Vol. 1062, pp. 209-216 (2005).

3. Michael Sulyok, Franz Berthiller, Rudolf Krska, Rainer Schuhmacher, “Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize,” Rapid Commun. Mass Spectrom., Vol. 20, pp. 2649-2659 (2006).

6. Zdenka Brenn-Struckhofova, Margit Cichna-Markl, Christina Bo1hm, Ebrahim Razzazi-Fazeli, “Selective Sample Cleanup by Reusable Sol-Gel Immunoaffinity Columns for Determination of Deoxynivalenol in Food and Feed Samples,” Anal. Chem., Vol. 79, pp. 710-717 (2007).

8. Ehrlich, K. C., and Cotty, P. J., “Variability in nitrogen regulation of aflatoxin production by Aspergillus flavus strains,” Appl. Microbiol. Biotechnol., Vol. 60, pp. 174-178 (2002).