New strategy for the determination of microcystins and diarrhetic shellfish poisoning (DSP) toxins, two potent phosphatases 1 and 2A inhibitors and tumor promoters

A new analytical strategy was established to improve the determination and identification performance during analyses of microcystins and diarrhetic shellfish poisoning (DSP) toxins in different matrices. Automated high performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algae and mussel tissue containing either microcystins or DSP toxins. The cleaned raw extracts are well suited for the direct determination of microcystins and DSP toxins by HPLC/MS. The analyses of cleaned raw extracts containing microcystin by HPLC and UV/diode array detection (DAD) revealed chromatograms without interfering peaks. Additionally, methods for the identification of unknown microcystins and those not available as standards were developed and established. The proposed strategy is exemplarily demonstrated for the analyses of a natural algae community from a lake in Slowakia and a naturally contaminated mussel from Portugal.     

Rapid clean-up and effective sample preparation procedure for unambiguous determination of the cyclic peptides microcystin and nodularin

Summary A new sample preparation strategy has been established to improve the identification and determination of nodularin and microcystins. The sample preparation consisted of enrichment of the analytes by solid phase extraction with C18 cartridges followed by clean-up of the enriched raw extracts by high performance size exclusion gel permeation chromatography. In contrast to established clean-up procedures based on polarity, related distribution of microcystins and nodularin in non-miscible phases (e. g. a C18 cartridge as stationary phase and a water-containing eluent as mobile phase) this strategy separates microcystins from interfering compounds by molecular size differences. The sample preparation procedure can be automated easily and was validated for both water samples as well as raw extracts of algal cells. The method was success-fully applied during an experiment with natural algae communities from the Baltic Sea to investigate the influence of different nutrient limitations on toxicity ofNodularia sp...     

Use of gel permeation chromatography for automatic and rapid extract clean-up for the determination of diarrhetic shellfish toxins (DSP) by liquid chromatography-mass spectrometry

Summary A new analytical technique was established to improve the performance of DSP toxin determination in different sample matrices. High performance size exclusion chromatography (gel permeation chromatography, SEC) was applied for the clean-up of raw extracts from algal cells and mussel tissue prior to the determination of DSP toxins by LC/MS. The proposed protocol can be performed totally automatically and enables fast and sensitive analysis of large sample numbers. The recovery of the entire method protocol (consisting of extraction, clean-up and LC/MS determination) was approximately 70% with good repeatability (standard deviation ranging from 1.9% to 5.0% in the concentration range analyzed).     

HPLC and HPCE analysis of microcystins RR, LR and YR present in cyanobacteria and water by using immunoaffinity extraction

Microcystins, which have their origin in species of cyanobacteria present in freshwaters, have recently been found to be important contaminants of the aquatic environment at trace levels. HPLC and HPCE with UV detection have been applied in the determination of such toxic compounds. Immunoaffinity chromatography for the selective extraction and clean-up of microcystins has been successfully applied to different matrices. Simple protocols for unambiguous determination of these toxins are presented and the immunoaffinity clean-up is compared with conventionally used solid phase extraction procedures. The development and optimisation of an on-line preconcentration procedure based on field amplified sample stacking for the analysis of microcystins by HPCE in the micellar electrokinetic chromatography mode is also described, using borate buffer with the anionic surfactant SDS, as separation electrolyte. Results indicate that sub-nanogram/gram content of microcystins can be detected in water samples, while sub-microgram/gram concentrations can be determined in algae samples.     

Simultaneous determination of toxins in algae and water samples by high-performance liquid chromatography with triple quadrupole mass spectrometry

A facile method based on liquid chromatography coupled with electrospray ionization tandem triple quadrupole mass spectrometry working in selected reaction monitoring mode has been established to analyze toxins in the algae and water samples. Twelve types of toxins (anatoxin, cylindrospermopsin, dinophysistoxin-1, nodularin, okadaic acid, microcystins) were efficiently separated under optimized liquid chromatography coupled with mass spectrometry conditions in the selected reaction monitoring mode. Correlation coefficients of the calibration curves, all felt in the range of 0.9958-0.9998, indicated good linearity. The detection limits of toxins in this method were all lower than 0.20 ng/mL and the quantification limits were in the range from 0.04 to 0.60 ng/mL. Except for anatoxin, cylindrospermopsin, and nodularin, the other toxins' recoveries varied from 55.45 to 140.85%. And the relative standard deviations of interday and intraday precision were at 8.61% (n = 5). The high-performance liquid chromatography (HPLC)/electrospray ionization (ESI)-mass spectrometery (MS) method was also successfully applied to analyze the algae and water samples. Owing to its exclusive selectivity and excellent sensitivity, the developed method is a tool for comprehensive analyses of the 12 types of toxins at nanogram levels.     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

Sensitive HPLC-fluorometric and HPLC-MS determination of diarrhetic shellfish poisoning (SP)-toxins as 4-bromomethyl-7-methoxycoumarin esters

 Extracts containing the diarrhetic shellfish poisoning (DSP) toxins okadaic acid (OA), dinophysistoxin-2 (DTX₂), and dinophysistoxin-1 (DTX₁) were purified on a silica gel cartridge and derivatized with 4-bromomethyl-7 methoxycoumarin (BrMmc). After pre-column derivatization the BrMmc derivatives of the DSP toxins were directly injected into an HPLC system, isocratically eluted, and quantified by fluorescence detection. The signals of the esters showed good linearity in the fluorescence detector within the examined contamination range of 0.03 mg DSP/kg to 2.5 mg DSP/kg. The detection limits for the DSP toxins as 7-Mmc esters were 0.04 ng (corresponding to 0.05 mg DSP/kg). The chromatographic conditions allow to couple the HPLC device with mass spectrometry. The method was tested with various mussel tissue samples. Received: 14 December 1995/Revised: 26 January 1996/Accepted: 30 January 1996     

Determination and confirmation of the amnesic shellfish poisoning toxin, domoic acid, in shellfish from Scotland by liquid chromatography and mass spectrometry

During 1998 and early 1999, shellfish samples from sites in Scotland were found to contain the amnesic shellfish poisoning toxin, domoic acid (DA). Two different techniques, liquid chromatography (LC) with UV diode-array detection and LC with mass spectrometric (MS) detection, were used to detect and confirm DA in shellfish extracts. The LC/UV method was validated for routine monitoring by recovery experiments on spiked mussel and scallop tissues with a certified mussel tissue used as reference material. Crude extracts of selected samples as well as extracts cleaned with strong anion exchange (SAX) were analyzed by both LC/UV and LC/MS. Good correlation (linear regression r2 = 0.996, slope = 0.93) between the 2 methods was found for cleaned extracts. Analyses of crude extracts by LC/UV produced false-positive results in 2 crab samples, whereas LC/MS analyses gave accurate results. It was concluded that LC/UV is a valid approach for routine monitoring of DA in shellfish when cleanup is performed with a SAX cartridge to prevent false positives. A variety of shellfish species were surveyed for DA content, including Pecten maximus (king scallops), Chlamys opercularis (queen scallop), Mytilus edulis (blue mussels), Cancer pugaris (crab), and Ensis ensis (razor fish). The highest concentration of DA was 105 microg/g in Pecten maximus.     

Phycotoxins in seafood--toxicological and chromatographic aspects.

Two typical clinical types of algae-related seafood poisoning have attracted medical and scientific attention: paralytic shellfish poisoning (PSP) and diarrhetic shellfish poisoning (DSP). Therefore, it became necessary to establish methods for the evaluation of possible hazards caused by contamination of seafood with these phycotoxins. Bioassays with mice or rats are the common methods for the determination of the toxin content of seafood. However, biological tests are not completely satisfactory because of a lack of sensitivity and pronounced variations. Additionally, there is growing opposition against animal testing. Therefore, many efforts have been undertaken to determine phycotoxins by chromatographic methods. PSP determination is mainly based on high-performance liquid chromatographic (HPLC) separation by ion-pair chromatography followed by postcolumn oxidation of the underivatized toxins in alkaline solution and fluorescence detection. HPLC methods for the determination of the DSP toxins okadaic acid (OA) and dinophysistoxin-1 (DTX-1) are characterized by precolumn derivatization with 9-anthryldiazomethane (ADAM) and/or 4-bromomethyl-7-methoxycoumarin (Br-Mmc), followed by chromatographic separation of the DSP esters formed and fluorescence detection. The chromatographic methods discussed in this review allow the rapid, sensitive and non-ambiguous determination of individual species of the two most important phycotoxins in seafood, PSP and DSP.     

Endocrine-disrupting nonylphenols - ultra-trace analysis and time-dependent trend in mussels from the German bight

A very sensitive and efficient analytical procedure is presented for the determination of 4-nonylphenols (NP) in blue mussels by use of off-line coupling of high-performance liquid chromatography (HPLC) and gas chromatography with mass spectrometric detection (GC-MS). Combined steam distillation and solvent extraction were used to extract the analytes from the mussel samples. Before quantification by GC-MS the raw extracts were purified by normal-phase HPLC. 4-n-Nonylphenol was used as internal standard. The detection limit was 15 ng NP absolute, calculated from the blank value. The method was applied to the determination of NP in blue mussel samples from the German North Sea sampled over a period of 10 years. Collection, homogenization, and storage of the mussels were performed according to the Standard Operating Procedures of the German Environmental Specimen Bank since 1985. The total NP concentrations in the mussels decreased significantly from 1985 (4 microgram kg (-1)) to 1995 (1.1 microgram kg (-1)).     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Determination of microcystins in blue-green algae, fish and water using liquid chromatography with ultraviolet detection after sample clean-up employing immunoaffinity chromatography

Anti-microcystin LR immunnoaffinity cartridges were evaluated for their ability to selectively remove microcystins from extracts of blue-green algae, fish and water samples for subsequent analysis by liquid chromatography with UV absorbance detection at 238 nm. Blue-green algae and fish samples were extracted with 75% methanol in water. A portion of the extract was diluted and passed through an immunoaffinity cartridge. Water samples were applied directly to the cartridge. The cartridge was rinsed with water and 25% methanol in water. The microcystins were eluted with 80% methanol in water containing 4% acetic acid. It was found that the cartridges were effective in isolating the microcystins from blue-green algae, fish and water samples, resulting in extracts that were clean enough to enable direct LC-UV detection down to approximately 0.03 microg/g in the blue-green algae and fish samples, and as low as 0.02 ng/ml for water samples. The cartridges were found to have a capacity of approximately 200 ng each for a mixture of microcystins RR, YR, LR and LA, or as much as 525-800 ng for individual compounds. Recoveries trough the complete analytical procedure ranged from 64 to 115% (all values) with an overall average of approximately 80% at spiking levels of 0.5-4.0 microg/g for the microcystins in blue-green algae. The average recoveries (n=8) from spiked (0.1-0.5 microg/g) fish samples were 73% for RR, 79% for YR, 81% for LR and 77% for LA, while from the spiked (2.0-0.04 ng/g) tap and river water samples (n=6), recoveries were 78% for RR, 86% for YR, 94% for LR and 89% for LA.     

Electrochemical detection of microcystins, cyanobacterial peptide hepatotoxins, following high-performance liquid chromatography

A novel amperometric HPLC detection method for the cyanobacterial (blue–green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were shown to originate in arginine and tyrosine residues of microcystins.     

Comparison of different fluorimetric HPLC methods for analysis of acidic polyether toxins in marine phytoplankton

The human toxic syndrome, diarrhetic shellfish poisoning (DSP), is caused by polyether toxins that are present in bivalve molluscs but originate from some species of marine phytoplankton. During the last few years different HPLC methods with fluorescence detection (FLD) have been proposed for analysis of marine toxins, including polyether toxins, in shellfish and phytoplankton. Several derivatization reagents have been proposed in the literature, with the aim of converting the acidic DSP toxins into their corresponding fluorescent derivatives. In this work we report results obtained from HPLC–FLD analysis of extracts from phytoplankton, including Dinophysis spp., harvested off the south-west coast of Ireland. Three different reagents were used for fluorescent derivatization: 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (BrDMEQ), 9-chloromethylanthracene (CA), and in situ 9-anthracenyldiazomethane (ADAM). Derivatization was performed under conditions previously optimised. The DSP derivatives were cleaned using different SPE procedures then analysed by HPLC–FLD. In this study, the use of BrDMEQ, CA, and in situ ADAM was compared in terms of sensitivity and selectivity. Evaluation of HPLC methods for analysis of DSP toxin derivatives was also conducted; the presence of okadaic acid (OA), dinophysistoxin-2 (DTX-2), and pectenotoxin-2 seco acids (PTX1SAs) was detected in the sample extracts studied.     

Improved method for the determination of anatoxin-a and two of its metabolites in blue-green algae using liquid chromatography with fluorescence detection

Anatoxin-a, a neurotoxin produced by blue-green algae (BGA) species, can cause death to exposed organisms. In North America, BGA are harvested and sold as food supplements, some of which contain elevated levels of other algal toxins, such as microcystins. Concern that elevated levels of anatoxin-a also may be present in BGA food supplements has led to the development of a simple method to determine the presence of anatoxin-a in BGA. Some researchers have successfully analyzed this compound using liquid chromatography with fluorescence detection by forming a fluorescent derivative with 4-fluoro-7-nitrobenzofurazan (NBD-F) in water and phytoplankton extracts. With this method, the background noise is high in BGA extracts due to the presence of co-extractives. Addition of o-phthaldialdehyde (OPA) and mercaptoethanol to the extract before addition of the NBD-F resulted in the successful removal of primary amines from the background noise when the NBD-F derivatives were detected with fluorescence. Improved chromatograms were obtained when extracts were cleaned up in this manner, leading to a lower detection limit (approximately 50 microg/kg) for anatoxin-a. The detection limits obtained for the 2 degradation products dihydroanatoxin-a and epoxyanatoxin-a in BGA extracts were similarly low (55 and 65 microg/kg, respectively).     

Fast screening and quantitation of microcystins in microalgae dietary supplement products and water by liquid chromatography coupled to time of flight mass spectrometry

Cyanobacteria, commonly called "blue-green algae", may accumulate in surface water supplies as "blooms" and may concentrate on the surface as blue-green "scums". Some species of cyanobacteria produce toxins and are of relevance to water supplies and to microalgae dietary supplements. To ensure the safety of drinking water and blue-green algae products, analyses are the only way to determine the presence or absence of toxins. This paper shows the use of ultra performance liquid chromatography (UPLC) coupled to orthogonal acceleration time of flight (TOF) mass spectrometry for the detection and quantitation of microcystins. The method presented is very sensitive, simple, fast, robust and did not require fastidious clean-up step. Limits of detection of 0.1 microg L(-1) in water and 0.1-0.2 microg g(-1) in microalgae samples were achieved. Method performances were satisfactory and appropriate for monitoring of water and dietary supplements. The method was applied in routine to samples taken from Swiss market or buy on internet website. Among 19 samples, six showed the presence of microcystins LR and LA at harmful levels.     

官厅水库蓝绿藻及藻毒素初步研究

聚苹果酸最初是由微生物学家在研究青霉菌的过程中发现的,聚苹果酸的人工合成由逐步聚合法开始,但由于苹果酸单体具有3种功能基,因此得到的是支化的聚苹果酸,随后人们改用苄酯基保护一端羧基的苹果酸单体,以DCC为缩合剂,进行缩聚,但这种方法只能得到聚苹果酸低聚物。     

红花水溶性多糖CTP的结构研究

微囊藻毒素（Microcystins,MCs）是由淡水蓝绿藻产生的一类最常见的环七肽缩氨酸肝毒素,它们通过抑制蛋白质磷酸酶的活性产生强烈的促肝癌作用,对人和其它生物产生较大威胁,人类长期饮用微量含有MCs的水,将导致原发性肝癌（PLA）,常见的MCs如MCLR,MCYR和MCRR的LD50值通常为50,70和600μg／kg（mouse,i．P．）。     

Comparison of liquid chromatography/mass spectrometry, ELISA, and phosphatase assay for the determination of microcystins in blue-green algae products

More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after sample cleanup using C18 solid-phase extraction. The results obtained by ELISA and LC-MS/MS agreed very well over a concentration range of about 0.5-35 microg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC-MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC-MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC-MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.     

Liquid chromatography-electrospray ionisation-mass spectrometry based method for the simultaneous determination of algal and cyanobacterial toxins in phytoplankton from marine waters and lakes followed by tentative structural elucidation of microcystins

A liquid chromatography (LC)-based method with mass spectrometric (MS) detection was developed for simultaneous determination of various algal and cyanobacterial toxins extracted from phytoplankton occurring world-wide in marine waters and lakes. The method enables quantification of saxitoxin, anatoxin-A, domoic acid, nodularin, microcystins, okadaic acid and dinophysistoxin-1 with a single chromatographic run. In addition, the applied chromatographic conditions allow isolation and identification of substances suspected to be "new" microcystins (cyclic peptides) by fraction collection, hydrolysis, derivatisation of resulting free amino acids with the modified chiral Marfey's reagent N-alpha-(2,4-dinitro-5-fluorophenyl)-L-valinamide (L-FDVA) and enantioselective analysis of the amino acid derivatives by LC-ESI-MS.