In the summer of 2012, over 750 dead and dying birds were observed at the Paul S. Sarbanes Ecosystem Restoration Project at Poplar Island, Maryland, USA (Chesapeake Bay). Clinical signs suggested avian botulism, but an ongoing dense Microcystis bloom was present in an impoundment on the island. Enzyme-linked immunosorbent assay (ELISA) analysis of a water sample indicated 6000 ng mL⁻¹ of microcystins (MCs). LC-UV/MS analysis confirmed the presence of MC-LR and a high concentration of an unknown MC congener (m/z 1037.5). The unknown MC was purified and confirmed to be [D-Leu¹]MC-LR using NMR spectroscopy, LC-HRMS and LC–MS², which slowly converted to [D-Leu¹,Glu(OMe)⁶]MC-LR during storage in MeOH. Lyophilized algal material from the bloom was further characterized using LC-HRMS and LC–MS² in combination with chemical derivatizations, and an additional 24 variants were detected, including MCs conjugated to Cys, GSH and γ-GluCys and their corresponding sulfoxides. Mallard (Anas platyrhynchos) livers were tested to confirm MC exposure. Two broad-specificity MC ELISAs and LC–MS² were used to measure free MCs, while ‘total’ MCs were estimated by both MMPB (3-methoxy-2-methyl-4-phenylbutyric acid) and thiol de-conjugation techniques. Free microcystins in the livers (63–112 ng g⁻¹) accounted for 33–41% of total microcystins detected by de-conjugation and MMPB techniques. Free [D-Leu¹]MC-LR was quantitated in tissues at 25–67 ng g⁻¹ (LC–MS²). The levels of microcystin varied based on analytical method used, highlighting the need to develop a comprehensive analysis strategy to elucidate the etiology of bird mortality events when microcystin-producing HABs are present.

2012年夏季，在美国马里兰州白杨树岛（切萨皮克湾）的保罗·萨班斯生态系统恢复项目中观察到超过750只死亡和垂死的鸟类。临床迹象表明禽肉中有肉毒杆菌中毒，但该岛上的蓄水库中却有密集的微囊藻开花。水样的酶联免疫吸附测定（ELISA）分析表明，有6000 ng mL ^-1的微囊藻毒素（MCs）。LC-UV / MS分析证实了MC-LR的存在和高浓度的未知MC同类物（m / z 1037.5）。使用NMR光谱，LC-HRMS和LC-MS ²纯化未知的MC，并确认为[D-Leu ¹ ] MC-LR ，然后缓慢转化为[D-Leu ¹，Glu（OMe）⁶在MeOH中储存期间] MC-LR。使用LC-HRMS和LC-MS ²结合化学衍生化进一步表征了大花中的冻干藻类物质，并检测到另外2​​4个变体，包括与Cys，GSH和γ-GluCys偶联的MC及其相应的亚砜。对绿头野鸭（Anas platyrhynchos）肝脏进行了测试以确认MC暴露。两种广泛特异性的MC ELISA和LC-MS ²用于测量游离MC，而“总” MC则通过MMPB（3-甲氧基-2-甲基-4-苯基丁酸）和巯基解偶联技术进行估算。肝脏中游离的微囊藻毒素（63–112 ng g ^-1）占通过去结合和MMPB技术检测到的总微囊藻毒素的33–41％。免费[D-Leu ¹] MC-LR在组织中的定量浓度为25-67 ng g ^-1（LC-MS ²）。微囊藻毒素的水平根据所使用的分析方法而有所不同，突出显示了需要开发一种综合分析策略来阐明存在微囊藻毒素的HAB时禽类死亡事件的病因。