Determination of Multiclass Cyanotoxins in Spirulina-Based Dietary Supplements Using a SLE-Tandem-SPE Procedure Followed by HILIC-MS/MS

M. del Olmo-Iruela, M. Aparicio-Muriana, Francisco J. Lara, A. García-Campaña
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引用次数: 2

Abstract

: Cyanobacteria are a diverse group of oxygenic photosynthetic prokaryotes, which are believed to be one of the oldest life forms on Earth. They live in a wide range of ecosystems and withstand extreme environmental conditions. An important proportion of cyanobacteria is known to be producers of harmful cyanotoxins, which are toxic secondary metabolites that can impact the ecosystem and human health. The oral route is one of the main ways whereby humans can be exposed to cyanotoxins. Therefore, the consumption of contaminated algae-based food supplements is becoming more relevant due to its upsurge, which underlines the importance of controlling these toxins in this kind of products. This work describes the simultaneous determination of seven cyanotoxins belonging to three different classes: the cyclic peptides microcystin-LR (MC-LR), microcystin-RR (MC-RR) and nodularin (NOD); the alkaloid anatoxin-a (ANA) and three non-protein amino acids isomers β -methylamine-L-alanine (BMAA), 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl)glycine (AEG). These have been determined in spirulina-derived food supplements using a novel solid–liquid extraction coupled with a solid phase extraction procedure for clean up and preconcentration (SLE-tandem-SPE) and analysis by hydrophilic interaction liquid chromatography with tandem mass spectrometry detection (HILIC-MS/MS). A SeQuant®Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC) column (EMD Millipore, Billerica, MA, USA) was employed to achieve the chromatographic separation in less than 12 min using water and acetonitrile, both acidified with 0.3% of formic acid, as mobile phase. Previously, an SLE was developed, using 4 mL of aqueous 5% formic acid to extract the most polar compounds, followed by 4 mL of 80% MeOH. Both extracts were combined and submitted to a tandem-SPE using mixed-mode cation exchange (MCX) and Strata-X cartridges. Elution from both cartridges was performed using 10% NH 3 · H 2 O in MeOH. Method validation was carried out in terms of linearity, limit of detection (LOD) and quantification (LOQ), recoveries, matrix effect and repeatability and intermediate precision. LOQs in the range of 50–300 µ g · kg − 1 and recoveries ranging between 64.2% and 102.9% with an associated relative standard deviation < 19.2% were achieved. Satisfactory precision was obtained with RSD values lower than 19.6% in all cases, with the exception of BMAA, which reported the highest RSD values, reaching 25.1%. The method was satisfactorily applied to determine the occurrence of cyanotoxins in blue green algae (BGA) dietary supplements. DAB was the most frequently detected cyanotoxin, at concentrations up to 2408 µ g · kg − 1 , and AEG was found in few samples at concentrations up to 194 µ g · kg − 1 . However, MC-LR and MC-RR were found in one sample at concentration levels higher than 5 mg · kg − 1 , which underlines the need to control these substances in these matrices.
以螺旋藻为基础的膳食补充剂中多类蓝藻毒素的串联固相萃取-液相色谱-质谱联用测定
蓝藻是一种不同的含氧光合原核生物,被认为是地球上最古老的生命形式之一。它们生活在广泛的生态系统中,能够承受极端的环境条件。众所周知,蓝藻菌的一个重要比例是有害的蓝藻毒素的生产者,这是有毒的次生代谢物,可以影响生态系统和人类健康。口服途径是人类接触到蓝藻毒素的主要途径之一。因此,受污染的藻类食品补充剂的消费由于其激增而变得更加相关,这强调了控制这类产品中这些毒素的重要性。本工作描述了同时测定属于3个不同类别的7种蓝藻毒素:环肽微囊藻毒素- lr (MC-LR),微囊藻毒素- rr (MC-RR)和结节素(NOD);生物碱anatoxin-a (ANA)和3种非蛋白质氨基酸异构体β -甲胺- l -丙氨酸(BMAA)、2,4-二氨基丁酸(DAB)和N-(2-氨基乙基)甘氨酸(AEG)。在螺旋藻衍生的食品补充剂中,使用一种新型的固液萃取结合固相萃取程序进行清理和预富集(SLE-tandem-SPE),并通过亲水相互作用液相色谱-串联质谱检测(HILIC-MS/MS)进行分析。采用SeQuant®两性离子亲水相互作用液相色谱(ZIC-HILIC)柱(EMD Millipore, Billerica, MA, USA),以0.3%甲酸酸化的水和乙腈为流动相,在不到12分钟的时间内实现色谱分离。以前,开发了SLE,使用4ml 5%甲酸水溶液提取最极性的化合物,然后使用4ml 80%的MeOH。将两种提取物组合并使用混合模式阳离子交换(MCX)和Strata-X墨盒提交到串联spe。用10%的nh3·h2o在MeOH中进行洗脱。从线性度、检出限(LOD)和定量限(LOQ)、回收率、基质效应、重复性和中间精密度等方面对方法进行验证。检出限为50 ~ 300µg·kg−1,加样回收率为64.2% ~ 102.9%,相关相对标准偏差< 19.2%。除BMAA的RSD值最高,达到25.1%外,所有病例的RSD值均低于19.6%,均获得满意的精度。该方法可用于测定蓝藻(BGA)膳食补充剂中蓝藻毒素的含量。DAB是最常见的蓝藻毒素,浓度高达2408µg·kg - 1, AEG在少量样品中发现,浓度高达194µg·kg - 1。然而,在一个样品中发现MC-LR和MC-RR的浓度水平高于5 mg·kg - 1,这强调了在这些基质中控制这些物质的必要性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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