Meisen Iris, Distler Ute, Müthing Johannes, Berkenkamp Stefan, Dreisewerd Klaus, Mathys Werner, Karch Helge, Mormann Michael
Institute for Hygiene, University of Münster, Robert-Koch-Strasse 41, D-48149 Münster, Germany.
Anal Chem. 2009 May 15;81(10):3858-66. doi: 10.1021/ac900217q.
Cyanobacteria are pathogenic prokaryotes and known for producing a high variety of cyclic hepatotoxic peptides in fresh and brackish water. Prominent members of these toxins are microcystin LR (MC LR) and nodularin (Nod), which are under suspicion to cause cancer. Various analytical methods have been reported for the detection of these cyclopeptides, and these are mainly based on liquid chromatography combined with mass spectrometric techniques. Here, we introduce a new approach based on the direct coupling of high-performance thin-layer chromatography (HPTLC) with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry (IR-MALDI-o-TOF MS) using the liquid matrix glycerol. The analysis of the cyclopeptides involves the application of three complementary methods: (i) HPTLC separation of MC LR and Nod, (ii) their detection and quantification by UV spectroscopy at lambda = 232 nm, and (iii) direct identification of separated analytes on the HPTLC plate by IR-MALDI-o-TOF MS. Calibration curves exhibited a linear relationship of amount of analyte applied for HPTLC and UV absorption (R(2) > 0.99). The limits of detection were 5 ng for UV spectroscopy and 3 ng for mass spectrometric analysis of individual peptides. This novel protocol greatly improves the sensitive determination of toxins from pathogenic cyanobacteria in complex water samples. It was successfully applied to the detection and quantification of MC LR and Nod in a spiked, processed environmental water sample.
蓝藻是致病性原核生物，以在淡水和微咸水中产生多种环状肝毒性肽而闻名。这些毒素的主要成员是微囊藻毒素LR（MC LR）和节球藻毒素（Nod），它们被怀疑会致癌。已经报道了多种检测这些环肽的分析方法，这些方法主要基于液相色谱与质谱技术的结合。在此，我们介绍一种基于高效薄层色谱（HPTLC）与红外基质辅助激光解吸/电离正交飞行时间质谱（IR-MALDI-o-TOF MS）直接耦合的新方法，使用液体基质甘油。环肽的分析涉及应用三种互补方法：（i）HPTLC分离MC LR和Nod，（ii）通过在λ = 232 nm处的紫外光谱对它们进行检测和定量，以及（iii）通过IR-MALDI-o-TOF MS直接鉴定HPTLC板上分离的分析物。校准曲线显示了HPTLC应用的分析物量与紫外吸收之间的线性关系（R² > 0.99）。紫外光谱的检测限为5 ng，单个肽的质谱分析检测限为3 ng。这种新方法大大提高了复杂水样中致病性蓝藻毒素的灵敏测定。它已成功应用于加标处理后的环境水样中MC LR和Nod的检测和定量。
