Direct Coupling of High-Performance Thin-Layer Chromatography with UV Spectroscopy and IR-MALDI Orthogonal TOF MS for the Analysis of Cyanobacterial Toxins

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.