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Polar marine toxins are more challenging to analyze by mass spectrometry-based methods than lipophilic marine toxins, which are now routinely measured in shellfish by multiclass reversed-phase liquid chromatography-tandem mass spectrometry (MS/MS) methods. Capillary electrophoresis (CE)-MS/MS is a technique that is well suited for the analysis of polar marine toxins, and has the potential of providing very high resolution separation. Here, we present a CE-MS/MS method developed, with use of a custom-built interface, for the sensitive multiclass analysis of paralytic shellfish toxins, tetrodotoxins, and domoic acid in seafood. A novel, highly acidic background electrolyte (5 M formic acid) was designed to maximize protonation of analytes and to allow a high degree of sample stacking to improve the limits of detection. The method was applied to a wide range of regulated and less common toxin analogues, and exhibited a high degree of selectivity between toxin isomers and matrix interference. The limits of detection in mussel tissue were 0.0052 mg/kg for tetrodotoxins, 0.160 mg/kg for domoic acid, and between 0.0018 and 0.120 mg/kg for paralytic shellfish toxins, all of which showed good linearity. Minimal ionization suppression was observed when the response from neat and mussel-matrix-matched standards was corrected with multiple internal standards. Analysis of shellfish matrix reference materials and spiked samples demonstrated good accuracy and precision. Finally, the method was transferred to a commercial CE-MS/MS system to demonstrate its widespread applicability for use in both R & D and routine regulatory settings. The approach of using a highly acidic background electrolyte is of broad interest, and can be considered generally applicable to simultaneous analysis of other classes of small, polar molecules with differing pK a values. Graphical abstract ᅟ.

The non-protein amino acid β-methylamino-L-alanine (BMAA) has been linked to neurodegenerative disease and reported throughout the environment. Proposed mechanisms of bioaccumulation, trophic transfer and chronic toxicity of BMAA rely on the hypothesis of protein misincorporation. Poorly selective methods for BMAA analysis have led to controversy. Here, a recently reported highly selective method for BMAA quantitation using hydrophilic interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS) is expanded to include proteinogenic amino acids from hydrolyzed biological samples. For BMAA quantitation, we present a double spiking isotope dilution approach using D 3 -BMAA and 13 C 15 N 2 -BMAA. These methods were applied to study release of BMAA during acid hydrolysis under a variety of conditions, revealing that the majority of BMAA can be extracted along with only a small proportion of protein. A time course hydrolysis of BMAA from mussel tissue was carried out to assess the recovery of BMAA during sample preparation. The majority of BMAA measured by typical methods was released before a significant proportion of protein was hydrolyzed. Little change was observed in protein hydrolysis beyond typical hydrolysis times but the concentration of BMAA increased linearly. These findings demonstrate protein misincorporation is not the predominant form of BMAA in cycad and shellfish.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by marine dinoflagellates that are responsible for paralytic shellfish poisoning (PSP) in humans. This work highlights our ongoing efforts to develop quantitative methods for PSTs using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). Compared with the commonly used method of liquid chromatography with post-column oxidation and fluorescence detection (LC-ox-FLD), HILIC-MS/MS has the potential of being more robust, sensitive and straightforward to operate, and provides unequivocal confirmation of toxin identity. The main driving force for the present work was the need for a complementary method to LC-ox-FLD to assign values to shellfish tissue matrix reference materials for PSTs. Method parameters that were optimized included LC mobile and stationary phases, electrospray ionization (ESI) conditions, and MS/MS detection parameters. The developed method has been used in the detection and identification of a wide range of PSTs including less common analogues and metabolites in a range of shellfish and algal samples. We have assessed the matrix effects of shellfish samples and have evaluated dilution, standard addition and matrix matched calibration as means of mitigating them. Validation on one LC-MS/MS system for nine common PST analogues (GTX1-4, dcGTX2&3, STX, NEO, and dcSTX) was completed using standard addition. The method was then transferred to a more sensitive LC-MS/MS system, expanded to include five more PSTs (C1&2, dcNEO and GTX5&6) and validated using matrix matched calibration. Limits of detection of the validated method ranged between 6 and 280 nmol/kg tissue using standard addition in extracts of blue mussels, with recoveries between 92 and 108%. Finally, this method was used in combination with the AOAC Official Method based on LC-ox-FLD to measure PSTs in a new mussel tissue matrix reference material.

Recent reports of the widespread occurrence of the neurotoxin β-N-methylamino-L-alanine (BMAA) in cyanobacteria and particularly seafood have raised concerns for public health. LC-MS/MS is currently the analytical method of choice for BMAA determinations but incomplete separation of isomeric and isobaric compounds, matrix suppression and conjugated forms are plausible limitations. In this study, capillary electrophoresis (CE) coupled with MS/MS has been developed as an alternative method for the quantitative determination of free BMAA. Using a bare fused silica capillary, a phosphate buffer (250 mM, pH 3.0) and UV detection, it was possible to separate BMAA from four isomers, but the limit of detection (LOD) of 0.25 μg mL -1 proved insufficient for analysis of typical samples. Coupling the CE to a triple quadrupole MS was accomplished using a custom sheath-flow interface. The best separation was achieved with a 5 M formic acid in water/acetonitrile (9:1) background electrolyte. Strong acid hydrolysis of lyophilized samples was used to release BMAA from conjugated forms. Field-amplified stacking after injection was achieved by lowering sample ionic strength with a cation-exchange cleanup procedure. Quantitation was accomplished using isotope dilution with deuterium-labelled BMAA as internal standard. An LOD for BMAA in solution of 0.8 ng mL -1 was attained, which was equivalent to 16 ng g -1 dry mass in samples using the specified extraction procedure. This was comparable with LC-MS/MS methods. The method displayed excellent resolution of amino acid isomers and had no interference from matrix components. The presence of BMAA in cycad, mussel and lobster samples was confirmed by CE-MS/MS, but not in an in-house cyanobacterial reference material, with quantitative results agreeing with those from LC-MS/MS. Graphical Abstract CE-MS separation and detection of BMAA, its isomers and the internal standard BMAA-d3.

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.