Anion-exchange Chromatography Method Research Articles

High-performance capillary electrophoresis to determine intact keratan sulfate and hyaluronic acid in animal origin chondroitin sulfate samples and food supplements.

Chondroitin sulfate is extracted from animal cartilaginous tissues and is commercialized as active principle against osteoarthritis. Its biological activity depends on its purity grade and could be altered by the presence of other glycosaminoglycans like keratan sulfate that could be contemporarily extracted from animal tissues or like hyaluronic acid that, instead, is added on purpose in food supplements. Although numerous methods are reported in literature for quality control analyses of chondroitin sulfate, few of them are able to detect other glycosaminoglycans. In this paper, for the first time, a new high-performance CE method was set up to quantify the chondroitin sulfate, the eventual keratan sulfate, and hyaluronic acid as intact chains: five chondroitin sulfate standards and 13 animal origin samples or food supplements from six different suppliers were analyzed. The new method was able to determine keratan sulfate similarly to a previously reported high-performance anion-exchange chromatography method, but in addition it showed the advantage to determine also the hyaluronic acid as never reported before.

Characterization and Identification of Allergen Protein in Shrimp Before and After Heated with SDS-PAGE Method

Food allergies are one of the most common allergies in Indonesian society. Generally, when children aged 5-6 years food allergies will disappear, except peanut allergies and allergies to seafood, such as fish, shellfish and crustaceans. This study aims to determine the pattern of separation of allergen proteins in shrimp using anion exchange column chromatography method and identify allergen proteins in shrimp using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Protein extraction from shrimp using Phosphate buffer saline (PBS) pH 7.2 and centrifuged at 10,000 rpm for 10 min at 4�C. Protein separation was carried out by anion-exchange column chromatography method, and the fraction obtained was measured at 280nm wavelength. The highest yield at absorbance was identified by using SDS-PAGE. Polyacrylamide gel electrophoresis was used to determine the protein profile and molecular weight of shrimp extract. Coloring of protein bands using silver staining. Data were analyzed descriptively based on the migration value of the sample protein bands compared to the marker protein band (Rf). The results of protein allergen profile analysis on shrimp using SDS-PAGE showed that the shrimp contained a protein band with a molecular weight of 37.77 kDa for cooked shrimp and 37.03 kDa for fresh shrimp.

Thioarsenite Detection and Implications for Arsenic Transport in Groundwater.

Arsenic toxicity and mobility in groundwater depend on its aqueous speciation. Uncertainty about the methods used for measuring arsenic speciation in sulfate-reducing environments hampers transport and fate analyses and the development of in situ remediation approaches for treating impacted aquifers. New anion-exchange chromatography methods linked to inductively coupled plasma mass spectrometry (ICP-MS) are presented that allow for sample/eluent pH matching. Sample/eluent pH matching is advantageous to prevent thioarsenic species transformation during chromatographic separation because species protonation states remain unaffected, hydroxyl-for-bisulfide ligand substitution is avoided, and oxidation of reduced arsenic species is minimized. We characterized model and natural solutions containing mixtures of arsenic oxyanions with dissolved sulfide and solutions derived from the dissolution of thioarsenite and thioarsenate solids. In sulfidic solutions containing arsenite, two thioarsenic species with S/As ratios of 2:1 and 3:1 were important over the pH range from 5.5 to 8.5. The 3:1 thioarsenic species dominated when disordered As2S3 dissolved into sulfide-containing solution at pH 5.4. Together with the preferential formation of arsenite following sample dilution, these data provide evidence for the formation and detection of thioarsenite species. This study helps resolve inconsistencies between spectroscopic and chromatographic evidence regarding the nature of arsenic in sulfidic waters.

Cracking Proteoform Complexity of Ovalbumin with Anion-Exchange Chromatography-High-Resolution Mass Spectrometry under Native Conditions.

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterization. Here, we report the development of a novel pH gradient-based anion-exchange chromatography method, which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterization of proteoforms at the intact protein level under native conditions. The analysis of four different proteins demonstrates outstanding chromatographic selectivity, while the mass spectra obtained are of excellent quality enabling the identification of proteoforms, including near isobaric variants, spanning 4 orders of magnitude in abundance. An in-depth analysis of ovalbumin from chicken egg white yields the identification and relative quantification of more than 150 different proteoforms, including fragmented and dimeric forms. More than 20 different ovalbumin charge variants together with their glycoform distributions are identified and quantified, many of which have not been reported previously.

At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification.

At‐line static light scattering and fluorescence monitoring allows direct in‐process tracking of fluorescent virus‐like particles. We have demonstrated this by coupling at‐line multi‐angle light scattering and fluorescence detectors to the downstream processing of enveloped virus‐like particles. Since light scattering intensity is directly proportional to particle concentration, our strategy allowed a swift identification of product containing fractions and rapid process development. Virus‐like particles containing the Human Immunodeficiency Virus‐1 Gag protein fused to the Green Fluorescence protein were produced in Human Embryonic Kidney 293 cells by transient transfection. A single‐column anion‐exchange chromatography method was used for direct capture and purification. The majority of host‐cell protein impurities passed through the column without binding. Virus‐like particles bound to the column were eluted by linear or step salt gradients. Particles recovered in the step gradient purification were characterized by nanoparticle tracking analysis, size exclusion chromatography coupled to multi‐angle light scattering and fluorescence detectors and transmission electron microscopy. A total recovery of 66% for the fluorescent particles was obtained with a 50% yield in the main product peak. Virus‐like particles were concentrated 17‐fold to final a concentration of 4.45 × 1010 particles/mL. Simple buffers and operation make this process suitable for large scale purposes.

A Validated Method for Detection and Quantitation of 2’Fucosyllactose in Infant Formula Matrices

Analytical methods to assess the content of free carbohydrates in solution range from simple tests of reductive power to combinations of chromatography and mass spectrometry. Soluble carbohydrates such as lactose, maltose, fructooligosaccharides, and galactooligosaccharides are commonly found in infant formulas either as sources of energy or soluble fibers. On the other hand, a rich repertoire of lactose-based carbohydrates occurs naturally in human milk. The advent of novel biosynthesis technologies resulted in the availability of human milk oligosaccharide structures that are being used as ingredients in infant formulas. Notably, 2’Fucosyllactose has been tested in preclinical and clinical studies to determine its safety and to explore its potential health benefits in the context of pediatric nutrition. Several chromatographic methods for the analysis of human milk oligosaccharides have been published and, the main challenge associated with 2’Fucosyllactose quantitation has been to improve its resolution from lactose, which is present at concentrations around 70 g/l in both, infant formula and human milk. We developed a high-performance anion exchange chromatography method to detect and quantify 2’ Fucosyllactose in the presence of lactose by expanding the elution time between these saccharides. We validated the analytical procedure which behaved linearly (average R=0.99951) at concentrations as low as 1.75 µg/ml (limit of quantitation) with an average limit of detection of 0.43 µg/ml.

Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories.Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73.To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises.By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

Starch digested product analysis by HPAEC reveals structural specificity of flavonoids in the inhibition of mammalian α-amylase and α-glucosidases

An accurate high-performance anion-exchange chromatography (HPAEC) method is presented to measure the inhibition property of flavonoids against mammalian starch digestive enzymes, because flavonoids interfere with commonly used 3,5-dinitrosalicylic acid (DNS) and glucose oxidase/peroxidase (GOPOD) methods. Eriodictyol, luteolin, and quercetin increased absorbance values (without substrate) in the DNS assay and, with substrate, either overestimated or underestimated values in the DNS and GOPOD assays. Using a direct HPAEC measurement method, flavonoids showed different inhibition properties against α-amylase and α-glucosidases, showing different inhibition constants (Ki) and mechanisms. The double bond between C2 and C3 on the C-ring of flavonoids appeared particularly important to inhibit α-amylase, while the hydroxyl group (OH) at C3 of the C-ring was related to inhibition of α-glucosidases. This study shows that direct measurement of starch digestion products by HPAEC should be used in inhibition studies, and provides insights into structure-function aspects of polyphenols in controlling starch digestion rate.

Generic anion-exchange chromatography method for analytical and preparative separation of nucleotides in the development and manufacture of drug substances

Nucleotides are among the most frequently used chemical building blocks in the research, development and manufacture of drug substances. They are composed of three highly polar subunit molecules (a nucleobase, a sugar, and at least one phosphate group), which makes their separation and analysis very challenging by conventional liquid chromatography techniques. Herein, we describe a simple, efficient, and cost-effective ion-exchange chromatography (IEC) method for the separation and purification of over 20 nucleotides. This method combines the use of a Tosoh TSKgel SuperQ-5P W resin in conjunction with a fully aqueous eluent profile (ammonium bicarbonate-based) that allows for a straightforward scale-up transition and convenient drying process with minimal environmental impact. This generic method was optimized using chromatography simulation software (ACD Labs/LC Simulator) and successfully applied to the preparative purification of multicomponent nucleotide mixtures using readily available Fast Protein Liquid Chromatography (FPLC) instrumentation. These IEC method conditions can be effectively applied as the starting point for method development and isolation of other highly polar nucleotide species beyond those investigated in this study.

Monosaccharide spectrum of arabinoxylane fraction of grain winter rye different genotypes

The analysis of the content and monosaccharide composition of the rye meal carbohydrate fraction of different varieties was carried out using high-performance anion-exchange chromatography method. The dominant component was glucose, in approximately equal proportions arabinose, xylose and galactose were detected. Our research has established partial degradation of arabinoxylans and starch at the beginning of water extraction at a temperature of 30оC that as a result of the work of exo-enzymes rye grain. Water extracts of meal contain from 52 to 74% carbohydrates in oligo - and polymeric form. Based on the content of xylose monosaccharide, rye Ogonek, Podarok and Marusenka are characterized as low pentosan, and Tatarskaya 1, Radon and Pamyati Kunakbaev as highly pentosan (9-11 and 14-16 mg of xylose/ 1 ml of extract, respectively).

One-step chromatographic method to purify α-lactalbumin from whey for nanotube synthesis purposes

A one-step anion-exchange chromatography method (NaCl gradient elution on a DEAE Sepharose™ Fast Flow gel column) was developed to purify α-lactalbumin (α-LA) from whey protein isolate. α-LA nearly 100% pure (based on the total protein content) was obtained with a yield of about 39%. Besides pure α-LA, which was the main objective of this work, highly pure β-lactoglobulin was also obtained with a yield of about 59%. The high purity of the obtained α-LA samples allowed its use to synthesise protein nanotubes with excellent gelation properties for their use as food thickeners and bioactive carriers. The samples’ purity degree obtained (based on the total protein content) was critical in the formation of proper nanotubes instead of random aggregates, which produced opaque and weak gels, less useful for food applications.

Cellular enzymatic anti-oxidants of fractionated mucus proteins from Eudrilus eugeniae (African night crawler) and Perionyx excavatus (Blue worm) in MC3T3.

To investigate SOD-liked, GPx-liked and cellular anti-oxidation activities of fractionated mucus proteins from Perionyx excavatus (Pe) and Eudrilus eugeniae (Ee) in the MC3T3 osteoblast precursor cell line. The crude mucus proteins were extracted from Pe and Ee and fractionated using the anion exchange chromatography method with FPLC. The fractionated proteins were studied to determine their SOD-liked and GPx-liked activities. The most efficient fractions were studied for cellular anti-oxidation activities in the MC3T3 cell line including scavenging, protecting and repairing conditions. The results showed that the highest SOD-liked activities of EeANX1 were found at 4.17 µg/ml and GPx-liked activities of EeANX4 were found at 5.26 µg/mL. EeANX1 had no cytotoxic effect on MC3T3 at 500 µg/mL, and the IC50 of EeANX4 was over 300 µg/mL. When both EeANX1 and EeANX4 were investigated for cellular anti-oxidation activity in MC3T3 cells at 20 µg/mL, the cellular superoxide and total ROS production of were significantly (p<0.05) lower than those of 250 µM pyocyanin (ROS inducer) in ROS scavenging, protecting and repairing conditions. The 20 µg/mL of EeANX1 and EeANX4 demonstrated increased SOD and GPx activities in MC3T3. Investigation of the scavenging, protection and repairing conditions of MC3T3 treated with 20 µg/mL each of EaANX1, EaANX4 and 250 µM pyocyanin demonstrated that the proteins had significantly lower SOD activities and higher GPx activities than the 250 µM pyocyanin. The EeANX1 and EeANX4 fractions demonstrate SOD-liked and GPx-liked activities, as well as cellular anti-oxidation activities. These fractions could be developed as a natural anti-oxidant. This research could provide benefit to the study of cellular anticancer.

A Highly Sensitive Anion Exchange Chromatography Method forMeasuring cGAS Activity in vitro.

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2'3'-cyclic GMP-AMP (2'3'-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatory factor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNA complex catalyzes the synthesis of 2'3'-cGAMP and the purpose of the protocol presented here is to measure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed to elucidate the relationship between dsDNA length and the level of cGAS activity. The method involves an in vitro reaction with low concentrations of cGAS and dsDNA followed by quantification of the reaction product using anion exchange chromatography. The low concentrations of cGAS and dsDNA and the high sensitivity of this assay is a key advantage when comparing different DNA fragments' ability to activate cGAS.

One pot synthesis of GDP-mannose by a multi-enzyme cascade for enzymatic assembly of lipid-linked oligosaccharides.

Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.

Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells

The nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a transcription factor in the regulation of many oxidative enzymes and efflux transporters critical for oxidative stress and cellular defense against xenobiotics. NRF2 is dysregulated in patient osteosarcoma (OS) tissues and correlates with therapeutic outcomes. Nevertheless, research on the NRF2 regulatory pathways and its potential as a therapeutic target is limited to the use of synthetic small interfering RNA (siRNA) carrying extensive artificial modifications. Herein, we report successful high-level expression of recombinant siRNA against NRF2 in Escherichia coli using our newly established noncoding RNA bioengineering technology, which was purified to >99% homogeneity using an anion-exchange fast protein liquid chromatography method. Bioengineered NRF2-siRNA was able to significantly knock down NRF2 mRNA and protein levels in human OS 143B and MG63 cells, and subsequently suppressed the expression of NRF2-regulated oxidative enzymes [heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1] and elevated intracellular levels of reactive oxygen species. In addition, recombinant NRF2-siRNA was effective to sensitize both 143B and MG63 cells to doxorubicin, cisplatin, and sorafenib, which was associated with significant downregulation of NRF2-targeted ATP-binding cassette (ABC) efflux transporters (ABCC3, ABCC4, and ABCG2). These findings support that targeting NRF2 signaling pathways may improve the sensitivity of cancer cells to chemotherapy, and bioengineered siRNA molecules should be added to current tools for related research.

The Determination of Sugars in Dairy Products: Development of a New Standard Method for the International Dairy Federation and the Internal Organization for Standardization.

A method using high-performance anion-exchange chromatography (HPAEC) with a pulsed amperometric detector (PAD) for the determination of mono- and disaccharides is described. The method was accepted by the International Dairy Federation and the Internal Organization for Standardization as a new work item for the determination of sugars in dairy matrixes, and the Milk and Milk Products technical committee of ISO/TC 34/SC 5 accepted the topic "Milk and milk products - Determination of the sugar contents - High-performance anion-exchange chromatographic method (HPAEC-PAD)" as a new work item. The proposed method consists of an aqueous ethanol extraction of the sugars in the dairy sample, followed by clarification with Carrez I and II reagents. The clarified filtrate is diluted and then directly introduced in the HPAEC-PAD system for quantification of the sugars. A single-laboratory validation of the proposed method has been scheduled for spring 2017.

Determination of Electroactive Organic Acids in Sugarcane Vinasse by High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection Using a Nickel Nanoparticle Modified Boron-Doped Diamond

Ethanol production process generates a huge quantity of vinasse. A suitable destination for this byproduct may be its utilization as source of chemical substances, by recovery within the biorefinery process. Vinasse is rich in organic acids, which present value-added due to their many industrial applications. In this context, the present work aimed the development of an anion-exchange chromatographic method with pulsed amperometric detection, using oxidized nickel nanoparticle modified boron-doped diamond electrode, to determine industrially interesting electroactive organic acids, such as lactic acid, malic acid, and tartaric acid, in sugarcane vinasse. The chromatographic separation was carried out in a CarboPac PA 1 column under gradient elution employing different proportions of 0.10 mol L–1 NaOH in 0.25 mol L–1 CH3COONa and deionized water. Under these conditions, lactic acid, malic acid, and tartaric acid were separated in 27 min. The limits of detection were 1.2 × 10–4 mol L–1 for lactic acid, 6.1 ...

The free and total myo-inositol contents of early lactation and seasonal bovine milk

A high performance anion-exchange chromatographic method employing pulsed amperometric detection was applied to the determination of endogenous free and total myo-inositol in bovine milk, for which there is limited information. The contents and trend variability of myo-inositol in milk from extensively pasture-fed cows during early lactation and across a production season were therefore evaluated. Free and total myo-inositol in seasonal milk were within the ranges of 2.3–4.5 mg 100 g−1 and 5.3–8.7 mg 100 g−1, respectively. This novel information will both improve understanding of the expression of innate myo-inositol in bovine milk, and provide manufacturers with information that can enhance formulation capability related to the production of cow's milk-based products

Thermotolerant alkaline protease enzyme from Bacillus licheniformis A10: purification, characterization, effects of surfactants and organic solvents

In this study, the extracellular thermostable alkaline protease out of A10 strain was purified 1.38-fold with 9.44% efficiency through the ammonium sulfate precipitation-dialysis and DE52 anion exchange chromatography methods. The molecular weight of the enzyme in question along with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined to be approximately 40.55 kDa, whereas the optimum pH and temperature ratings were identified as 9.0 and 70 °C, respectively. It was seen that the enzyme had remained stable between pH 7.5–10.5 range, protecting more than 90% of its activity in the wake of 1 h incubation at 60–70 °C. It was also observed that the enzyme enhanced its activity in the presence of Mg2+, Mn2+, K+, while Fe2+, Ni2+, Zn2+, Ag+ and Co2+ decreased the activity. Ca2+, however, did not cause any change in the activity. The enzyme was seen to have been totally inhibited by phenylmethylsulfonyl fluoride, therefore, proved to be a serine alkaline protease.

Identification of trace levels of selenomethionine and related organic selenium species in high-ionic-strength waters.

A new anion-exchange chromatographic separation method was used for the simultaneous speciation analysis of selenoamino acids and the more ubiquitous inorganic selenium oxyanions, selenite and selenate. For quantification, this separation was coupled to inductively coupled plasma-mass spectrometry to achieve an instrumental detection limit of 5 ng Se L(-1) for all species. This chromatographic method was also coupled to electrospray tandem mass spectrometry to observe the negative ion mode fragmentation of selenomethionine and one of its oxidation products. Low detection limits were achieved, which were similar to those obtained using inductively coupled plasma-mass spectrometry. An extensive preconcentration and cleanup procedure using cation-exchange solid-phase extraction was developed for the identification and quantification of trace levels of selenomethionine in environmental samples. Preconcentration factors of up to five were observed for selenomethionine, which in addition to the removal of high concentrations of sulphate and chloride from industrial process waters, allowed for an unambiguous analysis that would have been impossible otherwise. Following these methods, selenomethionine was identified at an original concentration of 3.2 ng Se L(-1) in samples of effluent collected at a coal-fired power plant's biological remediation site. It is the first time that this species has been identified in the environment, outside of a biological entity. Additionally, oxidation products of selenomethionine were identified in river water and laboratory algal culture samples. High-resolution mass spectrometry was employed to postulate the chemical structures of these species.