Korean Journal of Food Science and Technology (한국식품과학회지)

Korean Society of Food Science and Technology (한국식품과학회)
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Establishment of a Method for the Analysis of Diarrhetic Shellfish Poisoning by Liquid Chromatography-Tandem Mass Spectrometry

LC-MS/MS를 이용한 설사성패류독소의 분석조건 확립

  • Lee, Ka-Jeong (Food Safety Research Center, National Fisheries Research and Development Institute) ;
  • Suzuki, Toshiyuki (Biochemistry and Food Technology Division, National Research Institute of Fisheries Science, Fisheries Research Agency) ;
  • Kim, Poong-Ho (Food Safety Research Center, National Fisheries Research and Development Institute) ;
  • Oh, Eun-Gyoung (Food Safety Research Center, National Fisheries Research and Development Institute) ;
  • Song, Ki-Cheol (Food Safety Research Center, National Fisheries Research and Development Institute) ;
  • Kim, Ji-Hoe (Aquaculture Environment Research Institute, National Fisheries Research and Development Institute)
  • Published : 2009.08.31
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Abstract

To establish and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and accurate quantitation of diarrhetic shellfish poisoning (DSP) toxins, we compared the results from different mobile phases and columns used for their analysis and collision energies for MS/MS experiments. Clear peaks of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were obtained by using a mobile phase comprising aqueous acetonitrile containing 2 mM ammonium formate and 50 mM formic acid. The collision energies were optimized to facilitate the most sensitive detection for each toxin, namely, OA, DTX1, pectenotoxin-2 (PTX2), or yessotoxin (YTX). Further, the maximum ion response was obtained at a collision energy of 48 V for OA and DTX1. We compared the analytical performance of $C_8$ and $C_{18}$ columns. A wide range of toxins namely, OA, DTX1, PTX2, and YTX, except DTX3, were detected by both the columns. Although DTX3 was only detected by the $C_8$ column, we found that the $C_{18}$ column was also suitable for the quantitation of OA and DTX1 the toxins responsible for inducing diarrhea. The limit of detection of OA and DTX1 by the established LC-MS/MS conditions was 1 ng/g, and the limit of quantitation of the toxins under the same conditions was 3 ng/g. The process efficiencies were 91-118% for oysters (Crassostrea gigas) and 96-117% for mussels (Mytilus galloprovincialis) further, we observed no significant effect of matrix during the ionization process in LC-MS/MS. The comparison between mouse bioassay (MBA) and LC-MS/MS yielded varying results because low concentrations of OA and DTX1 were detected by LC-MS/MS in some shellfish samples, which provided positive results on MBA for DSP. The analysis time required by MBA for DSP analysis can be reduced by LC-MS/MS.

설사성패류독의 신속정밀 분석조건 확립을 위하여 LC-MS/MS를 사용하여 이동상, 분석용 column 및 collision energy 등을 변화시키면서 시험하였다. 50 mM formic acid와 2 mM ammonium formate가 함유된 acetonitrile 수용액을 이동상으로 사용하였을 때 OA와 DTX1이 검출되었다. Collision energy는 독소 성분에 따라 달리하는 것이 다성분 동시분석에 적합하였으며 OA와 DTX1 고유의 fragment ion들은 48 V 정도에서 최적의 intensity로 확인되었다. Column의 종류에 따라서는 $C_8$ column의 경우 OA, DTX1, DTX3, PTX2 및 YTX 모두 검출 가능하였으나 실제 검출 대상이 OA와 DTX1인 경우에는 일반적으로 사용되는 $C_{18}$ column도 적합한 것으로 확인되었다. 본 연구에서 확립한 LC-MS/MS 분석 조건의 검출한계는 OA와 DTX1 모두 1 ng/g, 정량한계는 각각 3 ng/g이었고, 표준독 성분을 첨가한 시료에서 process efficiency는 굴의 경우 91-118%, 진주담치에서는 96-117%이었고, matrix의 영향은 거의 없었다. 마우스 시험에서 양성을 나타낸 시료를 LCMS/MS법으로 분석한 결과, 일부 시료에서만 OA 및 DTX1이 검출되어 두 시험법의 독성은 일치하지 않았으며 LC-MS/MS법은 마우스 시험법보다 하루 이상 분석시간을 단축할 수 있었다.

Keywords

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