Hepatotoxic microcystins (MCs) are the most widespread class of cyanotoxins and the one that has most often been implicated in cyanobacterial toxicosis. One of the main challenges in studying and monitoring MCs is the great structural diversity within the class. The full chemical structure of the first MC was elucidated in the early 1980s and since then, the number of reported structural analogues has grown steadily and continues to do so, thanks largely to advances in analytical methodology. The structures of some of these analogues have been definitively elucidated after chemical isolation using a combination of techniques including nuclear magnetic resonance, amino acid analysis, and tandem mass spectrometry (MS/MS). Others have only been tentatively identified using liquid chromatography-MS/MS without chemical isolation. An understanding of the structural diversity of MCs, the genetic and environmental controls for this diversity and the impact of structure on toxicity are all essential to the ongoing study of MCs across several scientific disciplines. However, because of the diversity of MCs and the range of approaches that have been taken for characterizing them, comprehensive information on the state of knowledge in each of these areas can be challenging to gather. We have conducted an in-depth review of the literature surrounding the identification and toxicity of known MCs and present here a concise review of these topics. At present, at least 279 MCs have been reported and are tabulated here. Among these, about 20% (55 of 279) appear to be the result of chemical or biochemical transformations of MCs that can occur in the environment or during sample handling and extraction of cyanobacteria, including oxidation products, methyl esters, or post-biosynthetic metabolites. The toxicity of many MCs has also been studied using a range of different approaches and a great deal of variability can be observed between reported toxicities, even for the same congener. This review will help clarify the current state of knowledge on the structural diversity of MCs as a class and the impacts of structure on toxicity, as well as to identify gaps in knowledge that should be addressed in future research.

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Direct radical addition reactions at the C(8)-site of 2'-deoxyguanosine (dG) can afford C(8)-Ar-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons, and certain phenolic toxins. Such modified nucleobases are also highly fluorescent for sensing applications and possess useful electron transfer properties. The site-specific synthesis of oligonucleotides containing the C(8)-Ar-G adduct can be problematic. These lesions are sensitive to acids and oxidants that are commonly used in solid-phase DNA synthesis and are too bulky to be accepted as substrates for enzymatic synthesis by DNA polymerases. Using the Suzuki-Miyaura cross-coupling reaction, we have synthesized a number of C(8)-Ar-G-modified oligonucleotides (dimers, trimers, decamers, and a 15-mer) using a range of arylboronic acids. Good to excellent yields were obtained, and the reaction is insensitive to the nature of the bases flanking the convertible 8-Br-G nucleobase, as both pyrimidines and purines are tolerated. The impact of the C(8)-Ar-G lesion was also characterized by electrospray ionization tandem mass spectrometry, UV melting temperature analysis, circular dichroism, and fluorescence spectroscopy. The C(8)-Ar-G-modified oligonucleotides are expected to be useful substrates for diagnostic applications and understanding the biological impact of the C(8)-Ar-G lesion.

The neurotoxin β-N-methylamino-L-alanine (BMAA) has been reported in cyanobacteria and shellfish, raising concerns about widespread human exposure. However, inconsistent results for BMAA analysis have led to controversy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most appropriate method for analysis of BMAA, but the risk of interference from isomers, other sample components, and the electrospray background is still present. We have investigated differential mobility spectrometry (DMS) as an ion filter to improve selectivity in the hydrophilic interaction liquid chromatographic (HILIC)-MS/MS determination of BMAA. We obtained standards for two BMAA isomers not previously analyzed by HILIC-MS, β-amino-N-methylalanine and 3,4-diaminobutanoic acid, and the typically used 2,4-diaminobutanoic acid and N-(2-aminoethyl)glycine. DMS separation of BMAA from these isomers was achieved and optimized conditions were used to develop a sensitive and highly selective multidimensional HILIC-DMS-MS/MS method. This work revealed current technical limitations of DMS for trace quantitation, and practical solutions were implemented. Accurate control of low levels of DMS carrier gas modifier was essential, but required external metering. The linearity of our optimized method was excellent from 0.01 to 6 μmol L(-1). The instrumental LOD was 0.4 pg BMAA injected on-column and the estimated method LOD was 20 ng g(-1) dry weight for BMAA in sample matrix. The method was used to analyze cycad plant tissue, a cyanobacterial reference material, and mussel tissues, by use of isotope-dilution quantitation with deuterated BMAA. This confirmed the presence of BMAA and several of its isomers in cycad and mussel tissues, including commercially available mussel tissue reference materials certified for other biotoxins. Graphical Abstract Differential Mobility Spectrometry is used to increases the selectivity of BMAA analysis by HILIC-MS/MS.

Nearly a decade after first commercialization, high field asymmetric waveform ion mobility spectrometry (FAIMS) has yet to find its place in routine chemical analysis. Prototypes have been used to demonstrate the utility of this separation technique combined with mass spectrometry (MS). Unfortunately, first generation commercial FAIMS instruments have gone practically unused by early adopters. Here, we show this to be due to poor ion transmission in the FAIMS-MS source interface. We present simple instrumental modifications and optimization of experimental conditions to achieve good performance from the first generation commercial FAIMS device (the Ionalytics Selectra) coupled to a high resolution Q-TOF-MS. In combination with nanospray ionization, we demonstrate for the first time the nontarget analysis of urine by FAIMS with minimal sample preparation. We show the unique suitability of electrospray ionization (ESI)-FAIMS-MS for identification of low abundance species such as urinary biomarkers of damage of nucleic acids in a complex biological matrix. The elimination of electrospray noise and matrix components by FAIMS and the continuous flow of analytes through FAIMS for accurate and tandem mass analysis produce high quality spectral data suitable for structural identification of unknowns. These characteristics make ESI-FAIMS-MS ideal for nontarget identification, even when compared to high efficiency LC-ESI-MS.