Hlb 30 mg

Depending on the size of the tissue, samples were either processed separately, or as whole entrails. Samples of the same organ of all the individuals of the same species were pooled together. The extraction procedure was similar to the one described by Tokodi et al. [16 (link),27 (link)]. In short, samples were homogenized and freeze-dried. After freeze-drying, approximately 400 mg of homogenates were placed into a glass tube filled with 10 mL of 75% MeOH for cyanotoxin extraction overnight. Homogenization was then conducted for 30 s on ice, and the samples were ultrasonicated in a bath sonicator for 15 min, and additionally extracted with a probe sonicator (Bandelin Sonopuls HD 2070 micro-tip). Samples were then centrifuged for 10 min at 10,000× g. Ten milliliters of supernatant were then removed, and 5 mL of hexane were added. The lipid (hexane) layer was removed, and samples were concentrated by SPE (Waters Oasis HLB 30 mg) and eluted with 5 mL 90% MeOH. Two milliliters of each sample were then evaporated (50 °C, nitrogen flow), redissolved in 200 µL 25% MeOH and filtered (0.2 µm GHP Acrodisc 13; Pall Corporation, Port Washington, NY, USA) into inserts, thus ready for LC–MS/MS analysis.

Fish liver, gills, intestines, muscle, or whole entrails (if there was not enough material) of C. gibelio or A. brama from Fehérváricsurgó reservoir were analyzed for MCs by LC–MS/MS. Tissues were homogenized, then freeze-dried, and samples of the same organ of all the individuals of the same species were pooled together. Around 400 mg of freeze-dried fish tissue samples was placed into glass tubes, and 10 mL of 75% MeOH was added for extraction of cyanotoxins overnight. Probe homogenization was performed on ice for 30 s, ultrasonication in a bath sonicator for 15 min, and further extraction with a Bandelin Sonopuls HD 2070 micro-tip probe sonicator for 1 min (30% pulse and 30% energy). After centrifugation for 10 min at 10,000 × g, 5 mL of hexane to 10 mL of the obtained supernatants was added. The hexane (lipid) layer was discarded by a glass pipette. The samples were first diluted with water and then concentrated by SPE (Waters Oasis HLB 30 mg) and eluted with 5 mL 90% MeOH. Two milliliters of the samples was evaporated (50 ˚C nitrogen flow) in glass tubes and then redissolved in 200 µL 25% MeOH and filtered (0.2 µm GHP ACRODISC 13 PALL Corporation) into inserts. The fish tissue samples were then ready for LC–MS/MS analysis.