Md. Sagir Ahmed
University of Dhaka, Department
of Zoology, Advanced Fisheries and
DNA Barcoding Laboratory,
Dhaka 1000, Bangladesh
Thomas Krueger
University of Jena, Institute of
Nutrition, Department of Food
Chemistry, Dornburger Street 25,
07743 Jena, Germany.
Bernd Luckas
University of Jena, Institute of
Nutrition, Department of Food
Chemistry, Dornburger Street 25,
07743 Jena, Germany.
Anabaena, Cyanobacteria bloom, Microcystin
Brahmanbaria district.
Resource Development and Management
The study pond was 0.74 ha in size and located in Brahmanbaria district (23058' N latitude 91006' E longitude) 98 km east from Dhaka. Anabaena sp. bloom was initiated in the first week of April, 2008 and the highest cell density (90% Anabaena) was recorded on 14 April 2008. A portion of the concentrated samples were filtered through a 0.45 micro m glass fiber filter (Whatman GF/C, 47 mm diameter) and dried in an oven at 60-800C. Extraction: The GF/C filters were extracted with 2.0 ml of mixture water and methanol (50:50; v:v) per filter by ice-cooled sonication for 4 min with an ultrasonic probe GM 70 (Bandelin, Berlin, Germany) and subsequent treatment for 15 min in an ultrasonic bath. Extracts were centrifuged (10000 g, 15 min) and the supernatants were filtered using 0.22 micro m nylon syringe filters (Roth, Karlsruhe, Germany). The extracts were directly subjected to the liquid chromatography. Chemical analysis: The HPLC/UV determination of microcystins was carried out with some modification C18 column: Phenomenex prodigy, ODS (3), 250 x 4.6 mm, 5 ?m, mobil phases: acetonitrile /water/0.05% TFA). Detection of microcystins was done by the use of an UV detector (Shimadzu SPD-10AV; λ=238 nm). HPLC/MS and HPLC/MS-MS analysis were applied to ensure the identity of the toxin peaks in the chromatograms. Liquid Chromatography was performed with a PE Series 200 Quaternary Pump and a PE Series 200 autosampler (Perkin- Elmer, Shelton, CT, USA). The chromatographic separation was carried out on a reversed-phase column (Luna C18(2), 5 micro m 250 x 4.6 mm I.D., Phenomenex, Torrance) using gradient elution (0 min 20% B, 15-17 min 90% B, 18-30 min 20% B) with a flow rate of 1 mL min-1, throughout. Mobile phases consisted of 5 mM ammonium formate and 53 mM formic acid in (A) water and (B) water-acetonitrile (10:90, v:v), respectively. The HPLC was coupled by means of an electrospray interface to a single quadrupol mass spectrometer (API 150, PE Sciex Instruments, Canada) and additionally to a triple quadrupol mass spectrometer (API 365, PE Sciex Instruments, Canada). The detection was carried out in selected ion monitoring (SIM) mode using LC/MS and multiple reactions monitoring mode (MRM) using LC/MS-MS. Quantification: Since reference materials for desmethylated microcystins are not available commercially, determination of the concentrations of desmethyl-MCs, [D-Asp3, Dha7] MC-LR, and [Dha7] MC-LR, was performed using the standard calibration curves of MC-LR. This approach should be kept in mind looking at the toxin values given in this paper. Chemicals: Reference standards of Microcystin-RR, -LR, -YR, -LA, LF and –LW were purchased from Calbiochem/Novabiochem (La Jolla, CA, USA). Acetonitrile and methanol obtained from VWR (Leuven, Belgium) were HPLC grade. Water was purified to HPLC grade quality with a Millipore-Q RG Ultra Pure Water System (Millipore, Milford, USA). All chemicals were at least analytical grade.
International Journal of Fisheries and Aquatic Studies 2014; 2(3): 14-17 ISSN: 23 47-5129
Journal