Neurotoxin BMAA and its isomeric amino acids in cyanobacteria and cyanobacteria-based food supplements.

Cyanobacteria are photosynthetic microorganisms distributed globally in aquatic and terrestrial environments. They are also industrially cultivated to be used as dietary supplements, as they have a high nutritional value; however, they are also known to produce a wide range of toxic secondary metabolites, called cyanotoxins. BMAA (ß-methylamino-l-alanine) and its most common structural isomers, DAB (2,4-diaminobutyric acid) and AEG (N-2-aminoethylglycine) produced by cyanobacteria, are non-proteinogenic amino acids that have been associated with neurodegenerative diseases. A possible route of exposure to those amino acids is through consumption of food supplements based on cyanobacteria. The review critically discusses existing reports regarding the occurrence of BMAA, DAB and AEG in cyanobacteria and cyanobacteria-based food supplements. It is shown that inconsistencies in reported results could be attributed to performance of different methods of extraction and analysis applied and in ambiguities regarding determination of soluble and bound fractions of the compounds. The critical aspect of this review aims to grow awareness of human intake of neurotoxic amino acids, while results presented in literature concerning dietary supplements aim to promote further research, quality control as well as development of guidelines for cyanotoxins in food products.

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.

Determination of dencichine in Sanqi tablet by HILIC .

OBJECTIVE: To develop an HILIC method for determination of dencichine in Sanqi tablet and evaluate the quality of Sanqi tablet of different hatches from various manufactures in the market. METHOD: The chromatographic separation was conducted on a Thermo HILIC column (4.6 mm x 250 mm, 5 microm) kept at 25 degrees C with acetonitrile and 0.1% H3PO4 (60:40) as the mobile phase. The flow rate was set at 1 mL x min(-1) and the detection wavelength was set at 213 nm. RESULT: The contents of dencichine in Sanqi tablet ranged from 1.60 to 4.31 mg x g(-1). CONCLUSION: The well established method was successfully applied to determine dencichine in Sanqi tablet. The results demonstrated that this method was simple, accurate and could be applied for quality control of Sanqi as well as its associated preparations.

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.

[Content determination of dencichine in Panax Notoginseng by a reversed phase ion-pair chromatography].

To build a reversed phase ion-pair chromatography to determination content of Dencichine from Panax notoginseng. Using Tetrabutyl ammonium hydroxide ions by the combination of reagent and HPLC method without derivatization to test the content of dencichine directly. The optimum conditions of supersonic extraction were solid-to-liquid ratio 1: 20, Continuous ultrasonic extraction: twice, each time 15 minutes; 3,500 r · min⁻¹, then centrifuging 15 minutes. Dencichine in different age, place, part and the different Processing mode were examined. The method is simple with sound separation degree and stability, which can facilitate the determination of dencichine content directly and provide the basis in quality standard of raw material.

Environmental neurotoxins ß-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements.

Shark cartilage products are marketed as dietary supplements with claimed health benefits for animal and human use. Shark fin and cartilage products sold as extracts, dry powders and in capsules are marketed based on traditional Chinese medicine claims that it nourishes the blood, enhances appetite, and energizes multiple internal organs. Shark cartilage contains a mixture of chondroitin and glucosamine, a popular nutritional supplement ingested to improve cartilage function. Sharks are long-lived apex predators, that bioaccumulate environmental marine toxins and methylmercury from dietary exposures. We recently reported detection of the cyanobacterial toxin ß-N-methylamino-l-alanine (BMAA) in the fins of seven different species of sharks from South Florida coastal waters. Since BMAA has been linked to degenerative brain diseases, the consumption of shark products may pose a human risk for BMAA exposures. In this report, we tested sixteen commercial shark cartilage supplements for BMAA by high performance liquid chromatography (HPLC-FD) with fluorescence detection and ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC-MS/MS). Total mercury (Hg) levels were measured in the same shark cartilage products by cold vapor atomic fluorescence spectrometry (CVAFS). We report here that BMAA was detected in fifteen out of sixteen products with concentrations ranging from 86 to 265µg/g (dry weight). All of the shark fin products contained low concentrations of Hg. While Hg contamination is a known risk, the results of the present study demonstrate that shark cartilage products also may contain the neurotoxin BMAA. Although the neurotoxic potential of dietary exposure to BMAA is currently unknown, the results demonstrate that shark cartilage products may contain two environmental neurotoxins that have synergistic toxicities.

Homoarginine, ß-ODAP, and asparagine contents of grass pea landraces cultivated in Turkey.

The seeds of grass pea (Lathyrus sativus L.), a drought tolerant crop, were analysed for quantitative determination of the free amino acids ß-N-oxalyl-l-α,ß-diaminopropionic acid (ß-ODAP), homoarginine and asparagine by a simple and fast capillary electrophoretic method. In boric acid (80mM, pH 8.0) running buffer system, not only were α and ß-ODAP successfully separated, but also an efficient sample stacking was achieved during hydrodynamic sample introduction. The validated method was used for quantification of ß-ODAP, homoarginine and asparagine in seed extracts of 52 Lathyrus local landraces from various regions of Turkey and one released cultivar. The concentration ranges of amino acids were found as 0.21-1.27% (w/w) for homoarginine, 0.10-0.87% (w/w) for ß-ODAP and 0.006-0.47% (w/w) for asparagine. A positive correlation between homoarginine and ß-ODAP quantities in seeds of 53 Lathyrus local landraces was shown to exist (r(2)=0.649).

High-performance anion-exchange chromatography coupled with diode array detection for the determination of dencichine in Panax notoginseng and related species.

A high-performance anion-exchange chromatography coupled with diode array detection method was developed for the determination of dencichine in Panax notoginseng and related species. The analysis was performed on an Eprogen Synchropak WAX column (4.6 × 250 mm, 6 µm) with 50 mM NaH2 PO4 aqueous solution isocratic elution. The method was validated in terms of linearity, sensitivity, precision, stability, and accuracy. It was found that the calibration curve for dencichine showed good linearity (R(2) = 0.9999) within the test range. The LOD and LOQ were 0.77 and 3.06 ng, respectively. The RSD for intra- and interday repeatability was 0.2 and 0.5%, respectively. The test solution of dencichine is stable at least for three days at room temperature and for seven days at 4 °C. The mean recovery of dencichine was 102.0%. The established method was successfully applied to determine dencichine in the raw root of P. nogoginseng, P. ginseng, and P. quinquefolium as well as the steamed root of P. notoginseng. Compared with previous reports, this method is sensitive, selective, and accurate, which is helpful to evaluate the quality of P. notoginseng and related species.

LC-MS/MS determination of the isomeric neurotoxins BMAA (beta-N-methylamino-L-alanine) and DAB (2,4-diaminobutyric acid) in cyanobacteria and seeds of Cycas revoluta and Lathyrus latifolius.

Since diverse taxa of cyanobacteria has been linked to biosynthesis of BMAA, a controversy has arisen about the detection of neurotoxic amino acids in cyanobacteria. In this context, a novel LC-MS/MS method was developed for the unambiguous determination of beta-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB) in cyanobacteria and selected plant seeds. Both neurotoxic and non-proteinogenic amino acids were analyzed without derivatization considering the total concentration of the free and protein-bound form. The investigation of overall 62 cyanobacterial samples of worldwide origin by application of this method revealed the absence of BMAA, whereas seeds of Cycas revoluta contained 6.96 microg g(-1) of free BMAA. In contrast, the isomer DAB was confirmed in 16 cyanobacterial samples in concentrations of 0.07-0.83 microg g(-1),whereof one sample is distributed as nutritional supplement. In addition, seeds of Lathyrus latifolius contained 4.21 microg g(-1) of free DAB. Limits of detection were for BMAA<1.0 microg g(-1) in the cyanobacterial matrix and<0.14 microg g(-1) in angiosperm seeds. DAB exhibits higher sensitivities of <0.06 microg g(-1) in cyanobacteria and <0.008 microg g(-1) in angiosperm seeds. The highly specific analysis method with increased detection sensitivity eliminates the disadvantages of derivatization-based methods to be discussed.

Liquid chromatographic determination of the cyanobacterial toxin beta-n-methylamino-L-alanine in algae food supplements, freshwater fish, and bottled water.

Beta-N-Methylamino-L-alanine (BMAA) is a neurotoxin originally found in cycad seeds and now known to be produced by many species of freshwater and marine cyanobacteria. We developed a method for its determination in blue-green algae (BGA) food supplements, freshwater fish, and bottled water by using a strong cation-exchange, solid-phase extraction column for cleanup after 0.3 M trichloroacetic acid extraction of BGA supplements and fish. Bottled water was applied directly onto the solid-phase extraction column. For analysis of carbonated water, sonication and pH adjustment to 1.5 were needed. To determine protein-bound BMAA, the protein pellet left after extraction of the BGA supplement and fish was hydrolyzed by boiling with 6 M hydrochloric acid; BMAA was cleaned up on a C18 column and a strong cation-exchange, solid-phase extraction column. Determination of BMAA was by liquid chromatography of the fluorescent derivative formed with 9-fluorenylmethyl chloroformate. The method was validated by recovery experiments using spiking levels of 1.0 to 10 microg/g for BGA supplements, 0.5 to 5.0 microg/g for fish, and 0.002 microg/g for bottled water; mean recoveries were in the range of 67 to 89% for BGA supplements and fish, and 59 to 92% for bottled water. Recoveries of BMAA from spiked extracts of hydrolyzed protein from BGA supplements and fish ranged from 66 to 83%. The cleanup developed provides a useful method for surveying foods and supplements for BMAA and protein-bound BMAA.

Beta-methylamino-L-alanine analysis by liquid chromatography tandem mass spectrometry with iTRAQ as the derivative.

Amino acid BMMA is produced by cyanobacteria and has been linked to the development of neurodegenerative diseases. We developed a method for quantitative analysis of BMAA in biological samples and plant extracts. The method is utilizing iTRAQ and LC-MS/MS detection using multiple reaction monitoring mode. The method uses 50 microL of sample and has a limit of quantitation of 300 ng mL(-1), within-run run imprecision below 1%. Using this method we analyzed human serum samples, human cerebrospinal fluid samples and extract of the cycad seed. No BMAA could be detected in the human samples. Content of BMAA in the seed was 50 mg kg(-1).

Cyanobacteria (Nostoc commune) used as a dietary item in the Peruvian highlands produce the neurotoxic amino acid BMAA.

In the mountains of Peru, globular colonies of Nostoc commune (Nostocales) are collected in the highland lakes by the indigenous people, who call them llullucha. They are consumed locally, traded for maize, or sold, eventually entering the folk markets of Cusco and other neighboring cities. Throughout highland Peru, Nostoc commune is highly salient as a seasonal dietary item, being eaten alone, or in picante -- a local stew -- and is said to be highly nutritious. Nostoc commune has been known to produce unusual amino acids, including those of the mycosporine group, which possibly function to prevent UV damage. We analyzed 21 different Nostoc commune spherical colonies from 7 different market collections in the Cusco area for the presence of beta-N-methylamino-L-alanine (BMAA), a neurotoxic amino acid produced by diverse taxa of cyanobacteria, using four different analytical techniques (HPLC-FD, UPLC-UV, UPLC/MS, LC/MS/MS). We found using all four techniques that BMAA was present in the samples purchased in the Peruvian markets. Since BMAA has been putatively linked to neurodegenerative illness, it would be of interest to know if the occurrence of ALS, Alzheimer's, or Parkinson's Disease is greater among individuals who consume llullucha in Peru.

Smaragdicoccus niigatensis gen. nov., sp. nov., a novel member of the suborder Corynebacterineae.

A polyphasic taxonomic approach was applied to determine the taxonomic position of a hydrocarbon-degrading actinomycete, strain Hou_blueT, which was isolated from soil samples collected from an oil spring in Niigata, Japan. The results of 16S rRNA and gyrB gene sequence comparisons indicated that strain Hou_blueT represented a novel lineage in the suborder Corynebacterineae. Colonies were malachite green-like in colour on 1/10 trypticase soy agar and the cell morphology was coccoid in all growth phases. The cell-wall diamino acid and sugar indicated chemotype IV and variation A1gamma. The sugars of the peptidoglycan were glycolated. The polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside and some unspecified glycolipids. The organism contained two novel cyclic forms of menaquinone, smaragdiquinone A-8(H4, omega-cycl) and smaragdiquinone B-8(H4, dicycl). The major fatty acids were cis-9-18 : 1 (34.46 %) and 16 : 0 (25.1 %). Small amounts of 10-methyl-branched fatty acids were also present (10-methyl-17 : 0, 0.17 %), but not tuberculostearic acid (10-methyl-18 : 0), which has been shown to be present in all nocardiae. Gas-chromatographic analysis of the mycolic acid revealed a carbon-chain length of C43-C49. The DNA G+C content was 63.7 mol%. On the basis of phenotypic and phylogenetic distinctness, the organism is proposed to represent a novel genus and species, Smaragdicoccus niigatensis gen. nov., sp. nov., with the type strain Hou_blueT (=MBIC 06267T=DSM 44881T).

[Determination of dencichine in sanchi using liquid chromatography on porous graphitic carbon column].

A simple reversed-phase high performance liquid chromatographic method was developed for the determination of dencichine in sanchi on a porous graphitic carbon (PGC) column. The effects of different buffers, buffer concentrations, pH values of mobile phase and mobile phase compositions as well as column temperatures on the retention of dencichine were studied. The retention mechanism of dencichine on a porous graphitic carbon column seems to be mainly hydrophobic interaction according to the study. The chromatographic method developed was used to determine dencichine in sanchi. The calibration curve was linear in the range of about 0.22 - 4.4 microg (r = 0.999 9) and the recovery for the extract was 99.5% with the relative standard deviation of 2.2% (n = 9).

Analysis of dencichine in Panax notoginseng by gas chromatography-mass spectrometry with ethyl chloroformate derivatization.

Dencichine (beta-N-oxalyl-l-alpha,beta-diaminopropionic acid) is a haemostatic agent present in well-known traditional Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed) and cycad seeds. A method was developed for quantitative determination of the non-protein amino acid, dencichine, in plant samples of P. notoginseng and the adventitious roots directly from the explants of P. notoginseng after derivatization with ethyl chloroformate (ECF) by gas chromatography-mass spectrometry (GC-MS). l-2-chlorophenylalanine was used as an internal standard. Calibration curves were linear (r(2)=0.9988, n=6) in the range of 10-800 microg/ml for dencichine. Limit of detection and quantification for dencichine were 0.5 microg/ml and 2 microg/ml, respectively. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.

[Analysis of dencichine by HPLC with pre-column derivatization].

OBJECTIVE: To establish a reversed-phase high performance liquid chromatorgraphy (RP-HPLC) method for detecting the dencichine in Panax notoginseng extracts and drug preparations. METHOD: Dencichine was extracted with the borate buffer (pH 9. 18) and the clear supernatant was used for the derivatization. Pre-column derivatization was performed using 9-fluorenylmethyl chloroformate (FMOC) to form derivatives. The mobile phase consisted of methanol and 0. 05 mol x L( -1) NaH2 PO4 (48: 52) (pH adjusted to 7.4 with NaOH solution) in a flow rate of 1.0 mL m min(-1). The ultraviolet (UV) detection wavelength was set at 262 nm. RESULT: The linearity was demonstrated over a wide range of concentration from 1.76 mg L(-1) to 352 mg x L(-1) for dencichine. The detection limit was determined to be 60 microg x L(-1). The derivative was stable and the derivatization agent did not influence the measurement of dencichine. The average recovery rate was 95. 3% and the relative standard derivation (RSD) was 1. 7%. The method was used to determine dencichine in different P. notoginseng extracts and drug preparations. CONCLUSION: This method is simple, fast and sensitive, suitable for determining the dencichine in P. notoginseng extracts and drug preparations as well as for the study of the dencichine metabolism in vivo.

Hydrophilic interaction liquid chromatography with tandem mass spectrometry for the determination of underivatized dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) in Panax medicinal plant species.

Dencichine (beta-N-oxalyl-L-alpha,beta-diaminopropionic acid) is a haemostatic agent present in important Chinese medicinal herbs such as Panax notoginseng, as well as other Panax species. It is also a reported neurotoxic agent found in Lathyrus sativus (grass pea seed). A selective analytical method incorporating hydrophilic interaction chromatography with positive electrospray ionization tandem mass spectrometry (HILIC/ESI-MS/MS), for the analysis of dencichine in Panax plant species, was developed. Using multiple reaction monitoring (MRM) mode, underivatized dencichine, a small and highly polar compound, was selectively detected and quantified. The contents of dencichine in raw and steamed Panax notoginseng roots, 11 pairs of raw and steamed P. notoginseng herbal products, Panax ginseng roots, and Panax quinquefolium roots, were analyzed and compared. Optimal sensitivity of 0.3 ppm (detection limit) and 1.5 ppm (quantification limit) was achieved. The method was rapid (< or =5 min), with the HILIC peak eluting at about 1 min. Steamed P. notoginseng samples were found to contain less dencichine than the corresponding raw samples, and there were also differences among the three Panax species; raw P. ginseng and P. quinquefolium contained less dencichine than the raw P. notoginseng species. This rapid and specific method may be applied to the quantification of dencichine in complex medicinal plants and their products.

Chemical assessment of roots of Panax notoginseng in China: regional and seasonal variations in its active constituents.

Root of Panax notoginseng (Radix Notoginseng, Sanqi) is a well-known traditional Chinese medicine and is mainly cultivated in Wenshan of Yunnan, China. The active constituents include saponin, dencichine, flavonoid, and polysaccharide; however, the levels of these components vary in different geographical regions of growth and also show a seasonal variation. By using high-performance liquid chromatography and spectrophotometry, the contents of notoginsenoside R1, ginsenoside R(g1), R(b1), R(d), dencichine, flavonoid, and polysaccharide were determined and compared with Radix Notoginseng collected from different regions of growth in China, as well as from different seasons of harvest and market grades. Using the contents of these active constituents as markers, the best quality of Radix Notoginseng is found in the southwestern parts of Wenshan, and the best season for the harvest is September to October. In addition, the unseeded plants produced a better quality of Radix Notoginseng. The current results provide useful information for the quality control of Radix Notoginseng and its further development in establishing the good agriculture practice standard of P. notoginseng in China.

Analysis of neurotoxin 3-N-oxalyl-L-2,3-diaminopropionic acid and its alpha-isomer in Lathyrus sativus by high-performance liquid chromatography with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization.

A method was developed for the quantitative determination of the neurotoxic nonprotein amino acid, 3-N-oxalyl-L-2,3-diaminopropionic acid (beta-ODAP), and its nontoxic alpha-isomer, 2-N-oxalyl-L-2, 3-diaminopropionic acid (alpha-ODAP), in the plant samples of Lathyrus sativus after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) by reversed-phase high-performance liquid chromatography (HPLC). Hippuric acid was used as an internal standard. A linear response was recorded in the concentration rang 0.32-32 nmol with r > 0.999. The RP HPLC detection limit for both isomers is 1.8 ng. According to different experimental needs, a ternary gradient system can be used to determine toxin and other nonprotein amino acids. The RP HPLC method and a colorimetric method were compared for measuring ODAP.

Analysis of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid and homoarginine in Lathyrus sativus by capillary zone electrophoresis.

A simple capillary zone electrophoresis (CZE) method has been developed for the simultaneous quantitative determination of beta-N-oxalyl-L-alpha,beta-diaminopropionic acid (beta-ODAP) and homoarginine in Lathyrus sativus (LS; grass pea). A new Na2B4O7-Na2SO4 run buffer was used and the pH was 9.20, contents of beta-ODAP and homoarginine in crude extracts of LS plant material were determined with this method, the RSDs of peak areas of beta-ODAP and homoarginine were 2.62% and 3.61%, respectively. It was found that the equilibrium concentration ratio of alpha- and beta-ODAP decreased from 34.5/65.5 to 28.6/71.4 when the pH of the solution increased from pH 3.0 to pH 11.0.