NOAA - Great Lakes Environmental Research Laboratory
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Sample Collection
Samples were collected from 1 m depth using a submersible pump and filtered through a 90mm
Whatman GFC glass fiber filter and immediately placed at 0 degree for return to the lab.
To extract the toxins, the thawed filter was transferred to a 40-ml polypropylene centrifuge
tube containing 10-ml of a 50% methanol/water mixture containing 1% acetic acid and the filter
was extracted using ultrasound in three 20-sec bursts with 20 sec intervals between bursts to
allow for cooling down of the solution. This extraction technique gave a relatively high recovery
(recovery › 90%) for anatoxin-a as well as microcystins and PSP toxins as determined by HPLC-FD
and LCMS techniques. After extraction, the samples were centrifuged at 15,000 g for 10-15 min
in a Sorvall RC-5B Refrigerated Superspeed Centrifuge (DuPont Instruments, USA) to remove the
insoluble material. The supernatant was further passed through a 13 mm diameter, 45 mm pore
size syringe filter equipped with nylon membrane (Pall Life, USA) to remove small particulates.
The final sample solution was stored at –20°C for cyanobacterial toxins analysis.
Microcystin Analysis
Toxin filters were extracted by sonication in 10 mL of 50% aqueous methanol acidified to 1%
with acetic acid. Extracts were clarified by centrifugation at 27000 x g, followed by
filtration through a 0.45 µm nylon syringe filter and stored at -20°C. Microcystin concentration
was determined using the protein phosphatase inhibition assay (PPIA), modified from Carmichael
and An (1999), run in 96 well plates (Hotto et al., 2005). Microcystin variants were identified
from concentrated samples (10-20x) by HPLC (Ace 5 C18, 4.6 x 250 mm) with PDA and MS detectors
using a gradient of 30-70% acetonitrile to water, both acidified to 0.1% with trifluoroacetic
acid. Detection was at 239 nm (PDA) and by MS with electrospray ionization looking for molecular
ions between 800 to 1200 amu (Harada et al., 1996). Putative MC variants were compared to
standards, published molecular weights and UV spectra (Lawton and Edwards, 2001; Zurawell et al., 2005).
Anatoxin-a Analysis
Prior to analysis of water samples for anatoxin-a by high performance liquid chromatography
using fluorescence detection (HPLC-FD), 1-ml of extracted samples were placed in a 1.5 ml
microcentrifuge tube and dried in a SpeedVac. For analysis of anatoxin-a using HPLC-FD,
anatoxin-a standards or processed water samples were brought to 100 ul using sample buffer
(0.05 M pH 9.3 sodium borate buffer containing 50 ug internal standard, methyl pipecolinate,
Sigma-Aldrich, USA). The solution was derivatized by adding 50 ul of a 1 mg ml-1 NBD-F
(7-fluoro-4-nitrobenzofuranzan) dissolved in acetonitrile. The mixture was vortexed and
incubated in the dark at room temperature for 60 min. The reaction was terminated by addition
of 50 ul of a 1 M hydrochloric acid solution. The mixture was centrifuged (15,000 g for 5 min)
to settle particulate material and the supernatant was removed for anatoxin-a analysis.
Anatoxin-a and its degradation productions were analyzed by HPLC-FD according to James et al.
with modifications (James and Sherlock 1996; James et al. 1998). HPLC-FD analysis was performed
using an isocratic LC system equipped with a Water’s 510 HPLC pump, a Hitachi AS-2000 autosampler,
and a Shimadzu RF-535dual monochrometer fluorescence detector. ELAB software was used for
instrument control, data acquisition and processing. The system was equipped with a Dupont
Ace C18 column (4.6 x 250 mm, 5 µm; MAC-MOD Analytical, Inc., PA) with a C18 Phenomenex
guard column before the analytical column. A solvent flow rate of 0.8 ml min-1 was maintained
using a mobile phase of isocratic 55% acetonitrile in water for 20 min. The fluorescence detector
was set to an excitation wavelength of 470 nm and an emission wavelength of 530 nm. The limit of
detection (LOD) and the limit of quantification (LOQ) were presented in Table 3.2 and based on
20 ul standardized injection volume.
Gregory L Boyer
Professor of Biochemistry
Director, Great Lakes Research Consortium
State University of New York
College of Environmental Science and Forestry
Syracuse NY 13210
315-470-6825
email:
