CSS, under contract to NOAA, HAB Monitoring & Reference Branch, Stressor Detection and Impacts Division, National Centers for Coastal Ocean Science, NOAA National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA.
HAB Monitoring & Reference Branch, Stressor Detection and Impacts Division, National Centers for Coastal Ocean Science, NOAA National Ocean Service, 219 Fort Johnson Road, Charleston, SC 29412, USA.
J Chromatogr A. 2021 Feb 22;1639:461902. doi: 10.1016/j.chroma.2021.461902. Epub 2021 Jan 13.
Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.
在美国华盛顿州，应用欧盟海洋生物毒素参考实验室（EURLMB）推荐的 LC-MS/MS 标准方法测定脂溶性海洋生物毒素时，有 5 种贝类中的 4 种贝类的 okadaic 酸（OA）和 dinophysistoxin 1（DTX1）的提取效率较低（60-81%）。检测的毒素包括 OA 和包括酯在内的总 OA、DTX1、DTX2 和包括酯在内的总 DTX、azaspiracid 1、2 和 3（AZA1、AZA2 和 AZA3）、pectenotoxin 2（PTX2）和 yessotoxin（YTX）。采用与用于加标回收试验相同的对照样品制备基质匹配标准品，以评估毒素提取效率和样品净化程序。我们通过使用酸化甲醇提取或在 EURLMB 提取前将贝类匀浆在 70°C 下预煮 20 分钟来调整 EURLMB 提取方法。两种改良方法均显著提高了 OA 和 DTX1 的提取效率，并且包括预煮步骤后也显著提高了 YTX 的提取效率。然而，使用酸化甲醇提取时 YTX 的回收率较低，并且在包括预煮步骤时非贻贝类样品中 PTX2 的回收率较低。对水稀释的未水解样品进行正己烷洗涤，并将正己烷洗涤与固相萃取结合用于净化水解样品。通过改进样品净化，并结合 LC-MS/MS 调整，使用非基质匹配校准标准品，可在五个来自华盛顿州的贝类物种中定量测定美国食品药品监督管理局监管的毒素，结果具有可接受的准确性。
