Diamine Oxidase as a Therapeutic Enzyme: Study of Germination from Vegetal Sources and Investigation of the Presence of ß-N-Oxalyl-L-α,ß-diaminopropionic Acid (ß-ODAP) Using LC-MS/MS.

Vegetal diamine oxidase (vDAO), an enzyme proposed to relieve symptoms of histaminosis, shows better reactivity with histamine and aliphatic diamines, as well as higher enzymatic activity than DAO of animal origin. The objective of this study was to evaluate the enzyme activity of vDAO from germinating grains from Lathyrus sativus (grass pea) and Pisum sativum (pea), and to verify the presence of a neurotoxin, ß-N-Oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), in the crude extract obtained from their seedlings. A targeted liquid chromatography-multiple-reaction monitoring mass spectrometry method was developed and used to quantify ß-ODAP in the analysed extracts. An optimized sample preparation procedure, involving protein precipitation with acetonitrile followed by mixed-anion exchange solid-phase extraction, allowed for high sensitivity and good peak shape for ß-ODAP detection. The Lathyrus sativus extract exhibited the highest vDAO enzyme activity of the extracts, followed by the extract from pea cultivar Amarillo from the Crop Development Centre (CDC). The results have also shown that even though ß-ODAP was present in the crude extract from L. sativus, its content was far below the toxicity threshold (300 mg of ß-ODAP/kg body/day). CDC Amarillo showed 5000-fold less ß-ODAP than the undialysed L. sativus extract. It was concluded that both species can be considered as convenient sources of vDAO for potential therapeutic use.

Contrasting ß-ODAP content correlates with stress gene expression in Lathyrus cultivars.

Lathyrus sativus, commonly known as grass pea, is a nutrient-rich pulse crop with remarkable climate-resilient attributes. However, wide use of this nutritious crop is not adopted owing to the presence of a non-protein amino acid ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), which is neurotoxic if consumed in large quantities. We conducted a de novo transcriptomic profiling of two ODAP contrasting cultivars, Pusa-24 and its somaclonal variant Ratan, to understand the genetic changes leading to and associated with ß-ODAP levels. Differential gene expression analysis showed that a variety of genes are downregulated in low ß-ODAP cultivar Ratan and include genes involved in biotic/abiotic stress tolerance, redox metabolism, hormonal metabolism, and sucrose, and starch metabolism. Several genes related to chromatin remodeling are differentially expressed in cultivar Ratan. ß-ODAP biosynthetic genes in these cultivars showed differential upregulation upon stress. ODAP content of these cultivars varied differentially upon stress and development. Physiological experiments indicate reduced relative water content and perturbed abscisic acid levels in the low ODAP cultivar. Altogether, our results suggest that the low ODAP cultivar may have a reduced stress tolerance. The dataset provides insight into the biological role of ODAP and will be helpful for hypothesis-driven experiments to understand ODAP biosynthesis and regulation.

Microbial production of ectoine and hydroxyectoine as high-value chemicals.

Ectoine and hydroxyectoine as typical representatives of compatible solutes are not only essential for extremophiles to survive in extreme environments, but also widely used in cosmetic and medical industries. Ectoine was traditionally produced by Halomonas elongata through a "bacterial milking" process, of which the marked feature is using a high-salt medium to stimulate ectoine biosynthesis and then excreting ectoine into a low-salt medium by osmotic shock. The optimal hydroxyectoine production was achieved by optimizing the fermentation process of Halomonas salina. However, high-salinity broth exacerbates the corrosion to fermenters, and more importantly, brings a big challenge to the subsequent wastewater treatment. Therefore, increasing attention has been paid to reducing the salinity of the fermentation broth but without a sacrifice of ectoine/hydroxyectoine production. With the fast development of functional genomics and synthetic biology, quite a lot of progress on the bioproduction of ectoine/hydroxyectoine has been achieved in recent years. The importation and expression of an ectoine producing pathway in a non-halophilic chassis has so far achieved the highest titer of ectoine (~ 65 g/L), while rational flux-tuning of halophilic chassis represents a promising strategy for the next-generation of ectoine industrial production. However, efficient conversion of ectoine to hydroxyectoine, which could benefit from a clearer understanding of the ectoine hydroxylase, is still a challenge to date.

Transfer of the Neurotoxin ß-N-methylamino-l-alanine (BMAA) in the Agro-Aqua Cycle.

The neurotoxic non-protein amino acid ß-N-methylamino-l-alanine (BMAA) is connected to the development of neurodegenerative diseases. BMAA has been shown to accumulate in aquatic ecosystems, and filter-feeding molluscs seem particularly susceptible to BMAA accumulation. The blue mussels farmed along the Swedish coastline in the Baltic Sea are, due to their small size, exclusively used to produce feed for chicken and fish in the agro-aqua cycle. We have investigated the possible biotransfer of BMAA from mussels, via mussel-based feed, into chickens. Chickens were divided into two groups, the control and the treatment. BMAA was extracted from the muscle, liver, brain, and eye tissues in both chicken groups; a UPLC-MS/MS method was subsequently used to quantify BMAA. The results indicate detectable concentrations of BMAA in both chicken groups. However, the BMAA concentration in chicken was 5.65 times higher in the treatment group than the control group, with the highest concentration found in muscle tissue extracted from the treatment group chickens. These data suggest that there is a BMAA transfer route within the agro-aqua cycle, so further investigation is recommended before using mussel-based feed in the chicken industry.

Linking a rapid throughput plate-assay with high-sensitivity stable-isotope label LCMS quantification permits the identification and characterisation of low ß-L-ODAP grass pea lines.

BACKGROUND: Grass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin ß-L-oxalyl-2,3-diaminopropionic acid (ß-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties. RESULTS: A plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for ß-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled ß-L-ODAP) allowing accurate quantification of ß-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any ß-L-ODAP in these species. The LCMS method was also used to quantify ß-L-ODAP accurately in different tissues of grass pea. CONCLUSIONS: The plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and ß-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of ß-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for ß-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-ß-L-ODAP genotypes.

A systematic review of analytical methods for the detection and quantification of ß-N-methylamino-l-alanine (BMAA).

Neurodegenerative diseases are influenced by environmental factors such as exposure to toxins including the cyanotoxin ß-N-methylamino-l-alanine (BMAA) that can bioaccumulate in common food sources such as fish, mussels and crabs. Accurate and precise analytical methods are needed to detect and quantify BMAA to minimize human health risks. The objective of this review is to provide a comprehensive overview of the methods used for BMAA analysis from 2003 to 2019 and to evaluate the reported performance characteristics for each method to determine the consensus data for each analytical approach and different sample matrices. Detailed searches of the database Web of Science™ (WoS) were performed between August 21st, 2018 and April 5th, 2019. Eligible studies included analytical methods for the detection and quantification of BMAA in cyanobacteria and bioaccumulated BMAA in higher trophic levels, in phytoplankton and zooplankton and in human tissues and fluids. This systematic review has limitations in that only the English language literature is included and it did not include standard operating protocols nor any method validation data that have not been made public. We identified 148 eligible studies, of which a positive result for BMAA in one or more samples analyzed was reported in 84% (125 out of 148) of total studies, 57% of HILIC studies, 92% of RPLC studies and 71% of other studies. The largest discrepancy between different methods arose from the analysis of cyanobacteria samples, where BMAA was detected in 95% of RPLC studies but only in 25% of HILIC studies. Without sufficient published validation of each method's performance characteristics, it is difficult to establish each method as fit for purpose for each sample matrix. The importance of establishing methods as appropriate for their intended use is evidenced by the inconsistent reporting of BMAA across environmental samples, despite its prevalence in diverse ecosystems and food webs.

Vegetables cultivated with exposure to pure and naturally occurring ß-N-methylamino-L-alanine (BMAA) via irrigation.

Bioaccumulation and biomagnification of ß-N-methylamino-L-alanine (BMAA), a potent neurotoxin, has been demonstrated in various food webs. It is alarming as this intensification of BMAA will result in exposure to higher concentrations from a direct cyanobacterial source. As more food items are being identified as a source of BMAA and with the large variations in BMAA content, the aim of the present study was to evaluate BMAA uptake by, and accumulation in, two commonly consumed vegetables, Lactuca sativa and Allium fistulosum. Plants exposed to pure BMAA in controlled laboratory experiments, as well as vegetables naturally irrigated with water containing a BMAA producing cyanobacterial bloom were evaluated during growth and ripening. In the laboratory exposures, free BMAA was detected in both the edible ripe parts of L. sativa and A. fistulosum after 60 days of exposure to a total of 4.5 µg BMAA. However, in the bloom exposure samples no BMAA could be detected in the ripe vegetables of A. fistulosum, Cucurbita pepo, or Brassica rapa chinensis. The study emphasises the need to further screen items for BMAA to understand the human exposure risk as well as the difference between BMAA uptake patterns with free BMAA and that contained in cyanobacterial cells.

Prevalence of ß-methylamino-L-alanine (BMAA) and its isomers in freshwater cyanobacteria isolated from eastern Australia.

Environmental exposure to the amino acid ß-methylamino-L-alanine (BMAA) was linked to the high incidence of neurodegenerative disease first reported on the island of Guam in the 1940s and has more recently been implicated in an increased incidence of amyotrophic lateral sclerosis (ALS) in parts of the USA. BMAA has been shown to be produced by a range of cyanobacteria and some marine diatoms and dinoflagellates in different parts of the world. BMAA is commonly found with two of its constitutional isomers: 2,4- diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl) glycine (AEG). These isomers are thought to be co-produced by the same organisms that produce BMAA and MS/MS analysis following LC separation can add an additional level of specificity over LC-FL. Although the presence of BMAA and 2,4-DAB in surface scum samples from several sites in Australia has been reported, which Australian cyanobacterial species are capable of BMAA, 2,4-DAB and AEG production remains unknown. The aims of the present studies were to identify some of the cyanobacterial genera or species that can produce BMAA, 2,4-DAB and AEG in freshwater cyanobacteria blooms in eastern Australia. Eleven freshwater sites were sampled and from these, 19 single-species cyanobacterial cultures were established. Amino acids were extracted from cyanobacterial cultures and analysed using liquid chromatography-tandem mass spectrometry. BMAA was detected in 17 of the 19 isolates, 2,4-DAB was detected in all isolates, and AEG was detected in 18 of the 19 isolates, showing the prevalence of these amino acids in Australian freshwater cyanobacteria. Concentrations of all three isomers in Australian cyanobacteria were generally higher than the concentrations reported elsewhere. This study confirmed the presence of BMAA and its isomers in cyanobacteria isolated from eastern Australian freshwater systems, and determined which Australian cyanobacterial genera or species were capable of producing them when cultured under laboratory conditions.

An Improved HILIC HPLC-MS/MS Method for the Determination of ß-ODAP and Its α Isomer in Lathyrus sativus.

ß-N-Oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP) is a non-protein amino acid present in Lathyrus sativus (grass pea) and other Lathyrus species, in parallel with its nontoxic isomer, α-ODAP. When consuming grass pea for several months as staple food, ß-ODAP may cause neurolathyrism, a motor neuron degeneration syndrome. Therefore, the independent quantification of both ODAP isomers instead of only the total amount in grass pea allows the identification of less toxic varieties and the development of tools to support breeding for improving grass pea quality. In this work, a simple and fast HPLC-MS/MS method was developed without sample derivatization, using a hydrophilic interaction chromatography (HILIC) column and an isocratic gradient of eluents for 18 min, which allowed the determination of both α- and ß-ODAP. The proposed method was fully validated and applied to the determination of α- and ß-ODAP contents in a diverse collection of 107 grass pea accessions representative of the main grass pea-growing geographical regions in the world, with the prompt identification of contrasting accessions. ß-ODAP content in the analyzed grass pea samples ranged from 0.45 ± 0.02 to 6.04 ± 0.45 mg g-1. The moderate correlation found between α- and ß-ODAP contents (0.65) in this collection reinforces the importance of the independent quantification of both ODAP isomers.

A novel integrated strategy for the detection and quantification of the neurotoxin ß-N-methylamino-L-alanine in environmental samples.

We describe a set of new tools for the detection and quantification of ß-N-methylamino-L-alanine (BMAA) which includes a novel stable isotope-labeled BMAA standard (13C3,15N2) and a chip-based capillary electrophoresis mass spectrometry platform for separation and detection. Baseline resolution of BMAA from its potentially confounding structural isomers N-2-aminoethylglycine (AEG) and 2,4-diaminobutyric acid (2,4-DAB) is achieved using the chip-based CE-MS system in less than 1 min. Detection and linearity of response are demonstrated across > 3.5 orders of dynamic range using parallel reaction monitoring (PRM). The lower limit of detection and quantification were calculated for BMAA detection at 40 nM (4.8 ng/mL) and 400 nM (48 ng/mL), respectively. Finally, the strategy was applied to detect BMAA in seafood samples purchased at a local market in Raleigh, NC where their harvest location was known. BMAA was detected in a sea scallop sample. Because the BMAA/stable isotope-labeled 13C3,15N2-BMAA (SIL-BMAA) ratio in the scallop sample was below the limit of quantification, a semiquantitative analysis of BMAA content was carried out, and BMAA content was estimated to be approximately 820 ng BMAA/1 g of wet scallop tissue. Identification was verified by high mass measurement accuracy of precursor (< 5 ppm) and product ions (< 10 ppm), comigration with SIL-BMAA spike-in standard, and conservation of ion abundance ratios for product ions between BMAA and SIL-BMAA. Interestingly, BMAA was not identified in the free protein fraction but only detected after protein hydrolysis which suggests that BMAA is tightly bound by and/or incorporated into proteins. Graphical abstract Utilization of novel 13C3,15N2-BMAA and chip-based CE-MS/MS for detection and quantification of BMAA in environmental samples.

Optimization of the Determination Method for Dissolved Cyanobacterial Toxin BMAA in Natural Water.

There is a serious dispute on the existence of ß-N-methylamino-l-alanine (BMAA) in water, which is a neurotoxin that may cause amyotrophic lateral sclerosis/Parkinson's disease (ALS/PDC) and Alzheimer' disease. It is believed that a reliable and sensitive analytical method for the determination of BMAA is urgently required to resolve this dispute. In the present study, the solid phase extraction (SPE) procedure and the analytical method for dissolved BMAA in water were investigated and optimized. The results showed both derivatized and underivatized methods were qualified for the measurement of BMAA and its isomer in natural water, and the limit of detection and the precision of the two methods were comparable. Cartridge characteristics and SPE conditions could greatly affect the SPE performance, and the competition of natural organic matter is the primary factor causing the low recovery of BMAA, which was reduced from approximately 90% in pure water to 38.11% in natural water. The optimized SPE method for BMAA was a combination of rinsed SPE cartridges, controlled loading/elution rates and elution solution, evaporation at 55 °C, reconstitution of a solution mixture, and filtration by polyvinylidene fluoride membrane. This optimized method achieved > 88% recovery of BMAA in both algal solution and river water. The developed method can provide an efficient way to evaluate the actual concentration levels of BMAA in actual water environments and drinking water systems.

Analysis of BMAA enantiomers in cycads, cyanobacteria, and mammals: in vivo formation and toxicity of D-BMAA.

Chronic dietary exposure to the cyanobacterial toxin ß-N-methylamino-L-alanine (BMAA) triggers neuropathology in non-human primates, providing support for the theory that BMAA causes a fatal neurodegenerative illness among the indigenous Chamorro people of Guam. However, since there are two stereoisomers of BMAA, it is important to know if both can occur in nature, and if so, what role they might play in disease causation. As a first step, we analysed both BMAA enantiomers in cyanobacteria, cycads, and in mammals orally dosed with L-BMAA, to determine if enantiomeric changes could occur in vivo. BMAA in cyanobacteria and cycads was found only as the L-enantiomer. However, while the L-enantiomer in mammals was little changed after digestion, we detected a small pool of D-BMAA in the liver (12.5%) of mice and in the blood plasma of vervets (3.6%). Chiral analysis of cerebrospinal fluid of vervets and hindbrain of mice showed that the free BMAA in the central nervous system was the D-enantiomer. In vitro toxicity investigations with D-BMAA showed toxicity, mediated through AMPA rather than NMDA receptors. These findings raise important considerations concerning the neurotoxicity of BMAA and its relationship to neurodegenerative disease.

Quantitative determination of the neurotoxin ß-N-methylamino-L-alanine (BMAA) by capillary electrophoresis-tandem mass spectrometry.

Recent reports of the widespread occurrence of the neurotoxin ß-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.

A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction.

Exposure to ß-N-methylamino-l-alanine (BMAA) might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis, or directly followed by LC-MS/MS analysis) for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80%) and precise (mean relative standard deviation 10%), except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds) showed higher variation (relative standard deviation 21%-32%), implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%). Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

Grazing livestock are exposed to terrestrial cyanobacteria.

While toxins from aquatic cyanobacteria are a well-recognised cause of disease in birds and animals, exposure of grazing livestock to terrestrial cyanobacteria has not been described. This study identified terrestrial cyanobacteria, predominantly Phormidium spp., in the biofilm of plants from most livestock fields investigated. Lower numbers of other cyanobacteria, microalgae and fungi were present on many plants. Cyanobacterial 16S rDNA, predominantly from Phormidium spp., was detected in all samples tested, including 6 plant washings, 1 soil sample and ileal contents from 2 grazing horses. Further work was performed to test the hypothesis that ingestion of cyanotoxins contributes to the pathogenesis of some currently unexplained diseases of grazing horses, including equine grass sickness (EGS), equine motor neuron disease (EMND) and hepatopathy. Phormidium population density was significantly higher on EGS fields than on control fields. The cyanobacterial neurotoxic amino acid 2,4-diaminobutyric acid (DAB) was detected in plant washings from EGS fields, but worst case scenario estimations suggested the dose would be insufficient to cause disease. Neither DAB nor the cyanobacterial neurotoxins ß-N-methylamino-L-alanine and N-(2-aminoethyl) glycine were detected in neural tissue from 6 EGS horses, 2 EMND horses and 7 control horses. Phormidium was present in low numbers on plants where horses had unexplained hepatopathy. This study did not yield evidence linking known cyanotoxins with disease in grazing horses. However, further study is warranted to identify and quantify toxins produced by cyanobacteria on livestock fields, and determine whether, under appropriate conditions, known or unknown cyanotoxins contribute to currently unexplained diseases in grazing livestock.

Improved detection of ß-N-methylamino-L-alanine using N-hydroxysuccinimide ester of N-butylnicotinic acid for the localization of BMAA in blue mussels (Mytilus edulis).

ß-N-Methylamino-L-alanine (BMAA) is an important non-protein amino acid linked to neurodegenerative diseases, specifically amyotrophic lateral sclerosis (ALS). Because it can be transferred and bioaccumulated higher up the food chain, it poses significant public health concerns; thus, improved detection methods are of prime importance for the identification and management of these toxins. Here, we report the successful use of N-hydroxysuccinimide ester of N-butylnicotinic acid (C4-NA-NHS) for the efficient separation of BMAA from its isomers and higher sensitivity in detecting BMAA compared to the current method of choice using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization. Implementation of this efficient method allowed localization of BMAA in the non-visceral tissues of blue mussels, suggesting that more efficient depuration may be required to remove this toxin prior to consumption. This is a crucial method in establishing the absence or presence of the neurotoxic amino acid BMAA in food, environmental or biomedical samples.

Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and high-resolution mass spectrometry.

A new analytical method was developed for the detection of alkaloid cyanotoxins in harmful algal blooms. The detection of the nonproteinogenic amino acid ß-N-methylamino-L-alanine (BMAA) and two of its conformation isomers, 2,4-diaminobutyric acid (DAB) and N-(2-aminoethyl) glycine (AEG), as well as three alkaloid cyanotoxins, anatoxin-a (ANA-a), cylindrospermopsin (CYN), and saxitoxin (STX), is presented. The use of a chemical derivatization with dansyl chloride (DNS) allows easier separation with reversed phase liquid chromatography. Detection with high-resolution mass spectrometry (HRMS) with the Q-Exactive enables high selectivity with specific fragmentation as well as exact mass detection, reducing considerably the possibilities of isobaric interferences. Previous to analysis, a solid phase extraction (SPE) step is used for purification and preconcentration. After DNS derivatization, samples are submitted to ultra high-performance liquid chromatography coupled with heated electrospray ionisation and the Q-Exactive mass spectrometer (UHPLC-HESI-HRMS). With an internal calibration using isotopically-labeled DAB-D3, the method was validated with good linearity (R (2) > 0.998), and method limits of detection and quantification (MLD and MLQ) for target compounds ranged from 0.007 to 0.01 µg L(-1) and from 0.02 to 0.04 µg L(-1), respectively. Accuracy and within-day/between-days variation coefficients were below 15%. SPE recovery values ranged between 86 and 103%, and matrix effects recovery values ranged between 75 and 96%. The developed analytical method was successfully validated with 12 different lakes samples, and concentrations were found ranging between 0.009 and 0.3 µg L(-1) except for STX which was not found in any sample.

Selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS).

The neurotoxin ß-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, ß-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.

Biotransfer of ß-N-methylamino-L-alanine (BMAA) in a eutrophicated freshwater lake.

ß-N-Methylamino-L-alanine (BMAA), a neurotoxic non-protein amino acid, plays a significant role as an environmental risk factor in neurodegenerative diseases, such as amyotrophic lateral sclerosis. BMAA producers occur globally, colonizing almost all habitats and represent species from distinct phytoplanktonic groups, i.e., cyanobacteria, diatoms, and dinoflagellates. Bioaccumulation of BMAA in invertebrate and vertebrate organisms has also been registered around the globe. In the Baltic Sea, BMAA has been detected in several commercial fish species, raising the question of the bioaccumulation of BMAA in Swedish limnic systems. Here we find the presence of BMAA in water samples from Lake Finjasjön and identify its bioaccumulation patterns in both plankti-benthivorous and piscivorous fish, according to fish species, total weight, gender, and season of collection. For the first time, a large number of fish individuals were used in order to draw conclusions on BMAA bioaccumulation in a closed ecological community based on a statistical approach. We may, therefore, conclude that feeding patterns (plankti-benthivorous) and increased age of fish may lead to a higher tissue concentration of BMAA.

Determination of ß-N-methylamino-L-alanine, N-(2-aminoethyl)glycine, and 2,4-diaminobutyric acid in Food Products Containing Cyanobacteria by Ultra-Performance Liquid Chromatography and Tandem Mass Spectrometry: Single-Laboratory Validation.

A single-laboratory validation study was completed for the determination of ß-N-methylamino-L-alanine (BMAA), N-(2-aminoethyl)glycine (AEG), and 2,4-diaminobutyric acid (DAB) in bulk natural health product supplements purchased from a health food store in Canada. BMAA and its isomers were extracted with acid hydrolysis to free analytes from protein association. Acid was removed with the residue evaporated to dryness and reconstituted with derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor). Chromatographic separation and detection were achieved using RP ultra-performance LC coupled to a tandem mass spectrometer operated in multiple reaction monitoring mode. Data from biological samples were evaluated for precision and accuracy across different days to ensure repeatability. Accuracy was assessed by spike recovery of biological samples using varying amino acid concentrations, with an average recovery across all samples of 108.6%. The analytical range was found to be 764-0.746 ng/mL prior to derivatization, thereby providing a linear range compatible with potentially widely varying analyte concentrations in commercial health food products. Both the U. S. Food and Drug Administration (FDA) and U. S. Pharmacopeia definitions were evaluated for determining method limits, with the FDA approach found to be most suitable having an LOD of 0.187 ng/mL and LLOQ of 0.746 ng/mL. BMAA in the collected specimens was detected at concentrations lower than 1 µg/g, while AEG and DAB were found at concentrations as high as 100 µg/g. Finding these analytes, even at low concentrations, has potential public health significance and suggests a need to screen such products prior to distribution. The method described provides a rapid, accurate, and precise method to facilitate that screening process.