Sera samples (1 mL) were thawed equilibrated at roomtemperature for 15 minutes and vortex mixed. Exceptfor the blank, 25 μL of working solution of stanozolol-d3 (internal standard) was added to all samples. 50 μL of2 M formic acid were added, followed by 3 mL of iso-propanol/methanol/ (1:1, v/v) mixture and vortex mixedto release the protein bound analyte and to promoteprotein precipitation during a 15 minutes incubation at4°C. The resulting mixture was centrifuged at 3500 g for5 minutes at 4°C to remove the suspended matter, withsupernatants transferred to clean amber glass tubes forliquid-liquid extraction.
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