RESUMEN
The suitability of pressurized liquid extraction (PLE) of cyanotoxins from cells was investigated. The stability of cyanotoxins (MCYST-RR, MCYST-LR, and anatoxin-a) was evaluated at nine combinations of pressure and temperature (7, 10, and 14 MPa and 60 degrees C, 80 degrees C and 100 degrees C) using 75% (v/v) methanol in water (MeOH) as solvent. Additional experiments investigated the stability of cyanotoxins when water was used as solvent (at a pressure of 14 MPa and a temperature of 40 degrees C, 50 degrees C, 60 degrees C, 80 degrees C, or 100 degrees C). Results using 75% MeOH showed that the MCYST-RR and MCYST-LR were stable under the tested pressures up to 80 degrees C. At 100 degrees C MCYST recovery decreased by 10% to 17%. When water was used as the solvent, no differences in recovery were observed for MCYST-LR, whereas for MCYST-RR, maximum recovery was obtained at 60 degrees C, and degradation occurred at 100 degrees C. In contrast, anatoxin-a was labile under all experimental conditions; the best recoveries (ca. 50%) were obtained at 60 degrees C at the three pressures using 75% MeOH. However, only 17%-23% recovery was obtained with water extraction at all temperatures. The extraction of MCYST-LR and variants from cells (Microcystis aeruginosa, UTCC299) was studied using two solvents, 75% MeOH and 100% water, at 14 MPa and 60 degrees C and 100 degrees C. PLE extracts were compared with extracts obtained with 75% MeOH and ultrasonication. Complete extraction was achieved in both solvents in one 5-min cycle (at 100 degrees C). Although lower recovery was obtained using PLE (79%-105%), shorter extraction time and automation are advantageous over ultrasonication.
Asunto(s)
Cianobacterias/química , Inhibidores Enzimáticos/análisis , Péptidos Cíclicos/análisis , Técnicas de Química Analítica/métodos , Toxinas Marinas , Microcistinas , Presión , Solventes/química , TemperaturaRESUMEN
The efficiency of the in-source collision-induced dissociation (in-source CID) technique for the structural characterization of microcystins (MCYSTs) was evaluated. Microcystins that did not contain arginine underwent facile fragmentation to produce characteristic product ions at relatively low cone voltage and could be fully characterized based on their mass spectra. On the other hand, cyclic peptides possessing arginine residues, such as MCYST-RR, -LR, -YR and nodularin, were considerably more stable under in-source CID conditions and required higher cone voltage to induce fragmentation. This behaviour is explained in terms of the mobile proton model for peptide fragmentation that can be used as an indication for the presence of arginine when unknown microcystins are analyzed. In-source CID was applied to the characterization of microcystins released into water from a Microcystis aeruginosa culture (UTCC299) (UTCC: University of Toronto Culture Collection of Algae and Cyanobacteria). Six microcystins were detected in extracts from UTCC299: I, [D-Asp(3)]MCYST-LR; II, MCYST-LR; III, isomer of MCYST-LR; IV, isomer of methyl MCYST-LR; V, [D-Asp(3), Glu(OCH(3))(6)]MCYST-LR; and VI, [D-Glu(OCH(3))(6)]MCYST-LR. In-source CID provided mass spectral patterns similar to those obtained by CID in the collision cell of the mass spectrometer but was more sensitive for the analysis of microcystins.
Asunto(s)
Cromatografía Liquida/métodos , Microcystis/metabolismo , Péptidos Cíclicos/análisis , Péptidos Cíclicos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , MicrocistinasRESUMEN
Three different commercial standards of microcystin-RR were assessed for purity by the liquid chromatography coupled with electrospray ionization mass spectrometry (LC/ESI/MS) technique. Although the liquid chromatograms with photodiode array detector for each standard looked virtually identical, the analysis of corresponding mass spectra revealed that only one of them contained microcystin-RR per purity assay. The second standard was a mixture of microcystin-RR, and its demethyl variant identified as [Dha7]microcystin-RR, and the third one contained [Dha7]microcystin-RR only. We strongly recommend applying LC coupled with MS for purity assay of microcystin standards.
Asunto(s)
Péptidos Cíclicos/análisis , Anabaena/química , Indicadores y Reactivos , Toxinas Marinas , Microcistinas , Estándares de Referencia , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
A pilot study was conducted to provide preliminary data on the concentrations of perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorooctanesulfonamide (PFOSA) in the blood of Canadians. A set of 56 human serum samples was collected from non-occupationally exposed Canadians and analyzed by microbore HPLC-negative ion electrospray tandem mass spectrometry. PFOS was the main component of perfluorinated organic compounds (PFCs) and was detected in all 56 blood specimens at an average concentration of 28.8 ng mL(-1) and a range from 3.7 to 65.1 ng mL(-1). The concentration of PFOA was an order of magnitude lower than that of PFOS and was found only in 16 samples (29%) at concentrations above the limit of quantification (LOQ). PFOSA was not detected at levels above the method detection limit (MDL) in any of the samples. The levels of PFCs observed in the sample group of non-occupationally exposed humans in Canada were similar to the levels reported in a previous US study with a similar sample pool size. Two distinct PFOS isomers in human serum were identified by accurate mass determination.
Asunto(s)
Ácidos Alcanesulfónicos/sangre , Exposición a Riesgos Ambientales , Contaminantes Ambientales/sangre , Fluorocarburos/sangre , Adulto , Canadá , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Espectrometría de MasasRESUMEN
Microcystins (MCYSTs) were isolated from surface water using reusable immunoaffinity columns. Individual MCYST were determined by high performance liquid chromatography equipped with a photo-diode array detector (HPLC-PDA, 200-300 nm). Subsequent analysis of the samples by liquid chromatography-electrospray ionization mass spectrometry (LC-ESMS) provided molecular weight information, which was used to tentatively identify individual MCYST variants for which standards were not available. Results obtained using immunoaffinity columns (IAC)-HPLC-PDA were compared to those obtained using solid phase extraction (SPE) Oasis HLB-HPLC-PDA. This is the first report of the extraction of 15 microcystins and nodularin using immunoaffinity columns. Whereas previous reports demonstrates the use of IAC for four microcystins, we found that IAC selectively extracted the following microcystins: MCYST-RR, [D-Asp3]MCYST-RR, MCYST-YR, MCYST-LR, 3 MCYST-LR variants, MCYST-AR, MCYST-FR, MCYST-WR, MCYST-LA, MCYST-LA variant, the less polar microcystins such as MCYST-LF, MCYST-LW and nodularin. The IAC extracts were free of interferences which enabled better detection and identification of MCYSTs. Based on the amount loaded to the cartridges, the method detection limit was 10-14 ng when using IAC and 25 ng for SPE of each MCYST-RR, MCYST-YR and MCYST-LR. Reproducibilities expressed as relative standard deviation were 6-10% for SPE and 4-17% for IAC.
Asunto(s)
Toxinas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Péptidos Cíclicos/aislamiento & purificación , Agua/química , Anticuerpos , Cromatografía Líquida de Alta Presión/instrumentación , Cianobacterias/química , Toxinas Marinas/aislamiento & purificación , Microcistinas , Péptidos Cíclicos/química , Valores de ReferenciaRESUMEN
The inability to accurately assess exposure has been one of the major shortcomings of epidemiologic studies of disinfection by-products (DBPs) in drinking water. A number of contributing factors include a) limited information on the identity, occurrence, toxicity, and pharmacokinetics of the many DBPs that can be formed from chlorine, chloramine, ozone, and chlorine dioxide disinfection; b) the complex chemical interrelationships between DBPs and other parameters within a municipal water distribution system; and c) difficulties obtaining accurate and reliable information on personal activity and water consumption patterns. In May 2000, an international workshop was held to bring together various disciplines to develop better approaches for measuring DBP exposure for epidemiologic studies. The workshop reached consensus about the clear need to involve relevant disciplines (e.g., chemists, engineers, toxicologists, biostatisticians and epidemiologists) as partners in developing epidemiologic studies of DBPs in drinking water. The workshop concluded that greater collaboration of epidemiologists with water utilities and regulators should be encouraged in order to make regulatory monitoring data more useful for epidemiologic studies. Similarly, exposure classification categories in epidemiologic studies should be chosen to make results useful for regulatory or policy decision making.