L 2200 autosampler

The aggregability of platelets can be assessed by quantitative determination of ATP exocytosis [21 (link)]. WB samples containing low, physiological or high levels of albumin were centrifuged at 150 g for 12 min in order to obtain platelet rich plasma (PRP) samples. Platelet aggregation was induced by addition of collagen (2 μg mL^-1, final concentration) to 500 μL of the PRP samples. After two minutes, the PRP samples were centrifuged at 1,500 g for two minutes and proteins in the supernatant were precipitated with 0.4 M perchloric acid. After centrifugation at 12,000 g 100 μL of the supernatant were neutralized by addition of 10–12 μL of 2 M K₂CO₃ at 4°C. The supernatant obtained after centrifugation was used for HPLC analysis (injection volume: 40 μL). Separation of adenine nucleotides was performed on a Hypersil ODS column (5 μm, 250 × 4 mm I.D., equipped with a precolumn; Thermo Electron Corp. Runcorn, Cheshire, UK) using a L-2200 autosampler, two L-2130 HTA pumps, and a L-2450 diode array detector (all VWR International, West Chester, PA, USA) as previously described [22 (link)]. Detector signals (absorbance at 254 nm) were recorded and the program EZchrom Elite (VWR) was used for data acquisition and analysis.

The concentrations of both test compounds in serum and tissue homogenates were measured by a reverse-phase high-performance liquid chromatography method with ultraviolet detection (HPLC/UV). Samples were separated on a Supelcosil LC-PCN column (250 × 4.6 mm, 5 μm particle size) with an integrated pre-column (Sigma-Aldrich, Bellefonte, PA, USA). The mobile phase consisted of methanol/10 mM potassium dihydrogen phosphate buffer, pH 4.6/acetonitrile (51/40/9, v/v/v). The isocratic separation was performed at 1.0 mL/min constant flow-rate with column temperature maintained at 38 °C. The detection wavelength was set at 206 nm for 2 and at 215 nm for 3. The HPLC system consisted of a Hitachi-Elite LaChrom L-2130 pump, with an ultraviolet-visible (UV-Vis) detector (L-2400), a LaChrom L-2300 column oven, and an L-2200 autosampler (VWR, Darmstadt, Germany). EZChrome Elite v. 3.3.2 software (VWR, Darmstadt, Germany) was used for data acquisition.

In order to determine the soluble fraction of the investigated compound in biological media, HPLC–DAD analysis was performed. VWR Hitachi HPLC–DAD systems (containing the L-2130 HTA-pump, L-2130 degasser, L-2200 autosampler, L-2300 column oven, L-2450 diode array-detector and the EZChrom Elite software, VWR, Darmstadt, Germany) was used. The separation was performed on Gemini C₁₈ column (150 × 4.6 mm I.D., 5 μm pore size, Phenomenex Inc., Torrance, CA). A mixture of A–ACN and B–H₂O was used as a mobile phase (gradient conditions, 30 % A in 0 min, 60 % A in 5 min and 30 % A in 10 min). The injection volume of the sample was 50 μL. A detection wavelength of 300 nm was used for the quantification. The analysis time was 15 min.
The concentrations of stock solution of FLU and FEN in media solutions used in four ecotoxicity tests were as follows: 1 mg L⁻¹ for L. minor and S. vacuolatus (the solution consisted 0.2 % DMSO), 0.05 mg L⁻¹ (0.01 % DMSO) for FEN and 0.075 mg L⁻¹ (0.015 % DMSO) for FLU for D. magna and 0.3 mg L⁻¹ for V. fischeri (0.06 % DMSO). In order to calculate the bioavailability, the peak area of the signals obtained from the analysis of the above-mentioned mixtures were compared to the peak area of the signals obtained from the analysis of the same concentrations of FLU and FEN prepared in ACN.