Background

Microcystins (MCs) and nodularins (NODs) produced by cyanobacteria occur in ambient freshwaters and across the freshwater-marine continuum, and pose health threats through drinking and recreational waters, as well as food resources. Approximately 300 MC and NOD toxins have been published, but less than 15 of them are commercially available as toxin standards. Our aim herein was to rapidly identify and quantify all toxin congeners, including those without standards, in water samples even at low abundance by reversed-phase solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide insights into toxin levels and potential toxicity.

Results

Trizma instead of acid was used as an ion pairing reagent to increase retention of MCs without arginine residues on Strata X SPE. Toxin elution from Oasis HLB SPE was complicated by their hydrophilic and lipophilic interactions with HLB sorbent. Three stable isotope-labeled (SIL) toxin standards representing MCs carrying 2, 1, and 0 arginine residues served as internal standards (IS) for toxin determination and evaluation of cyanobacterial cell lysis methods. Average recoveries of toxin standards in lake water and seawater using the HLB sorbent for validation ranged from 90 to 109% except MC-WR & LW (71 to 87%) with detection limits from 1.3 to 23.7 ng L^-1. Doubly charged protonated toxin molecular ions were employed as precursors for tandem MS to screen and identify toxin congeners using a hybrid triple quadrupole linear ion trap MS. More than 30 (4 new) MCs were detected in Microcystis aeruginosa strain LE-3 culture.

Significance

This is the first report to explain the issues with and possible mechanisms for SPE extraction of MCs from water, to use SIL-IS to evaluate methods for lysing cyanobacterial cells for MC release, and to show that doubly rather than singly charged toxin molecular ions as MS/MS precursors enabled efficient screening, identification, and quantification of all toxins at low levels. A MC with homoalanine residue at position 1 was reported for the first time.

中文翻译：

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通过液相色谱-质谱法测定环境淡水和海水中的微囊藻毒素和结节蛋白，包括毒素筛查和鉴定

 背景

蓝藻产生的微囊藻毒素 （MC） 和结节菌素 （NOD） 存在于环境淡水中以及淡水-海洋连续体中，并通过饮用水和娱乐水域以及食物资源对健康构成威胁。已经发表了大约 300 种 MC 和 NOD 毒素，但其中只有不到 15 种作为毒素标准品在市场上出售。本文的目的是通过反相固相萃取 （SPE）-液相色谱-串联质谱 （LC-MS/MS） 快速鉴定和定量水样中所有毒素同系物，包括无标准品的同系物，即使是低丰度的毒素，以深入了解毒素水平和潜在毒性。

 结果

使用 Trizma 代替酸作为离子对试剂，以提高 MC 在 Strata X SPE 上无精氨酸残留的保留性。Oasis HLB SPE 的毒素洗脱因与 HLB 吸附剂的亲水和亲脂相互作用而变得复杂。三种稳定同位素标记 （SIL） 毒素标准品代表携带 2、1 和 0 精氨酸残基的 MC，可用作毒素测定和蓝藻细胞裂解法评估的内标 （IS）。使用HLB吸附剂进行验证的湖泊水和海水中毒素标准的平均回收率在90%至109%之间，但MC-WR和LW（71至87%）的检测限为1.3至23.7 ng ^L-1。采用双电荷质子化毒素分子离子作为串联 MS 的前体，使用杂化三重四极杆线性离子阱 MS 筛选和鉴定毒素同系物。在铜绿微囊藻菌株 LE-3 培养物中检测到 30 多种 （4 种新） MCs。

 意义

这是第一份解释 SPE 从水中提取 MC 的问题和可能机制的报告，使用 SIL-IS 评估裂解蓝藻细胞以释放 MC 的方法，并表明双电荷而不是单电荷毒素分子离子作为 MS/MS 前体能够高效筛选、鉴定和定量所有低水平毒素。首次报道了在位置 1 处具有高异丙氨酸残基的 MC。