2.4.1. Liquid–Liquid Extraction

An aliquot (10 μL) of the analyte working solution and an aliquot (10 μL) of the IS working solution (5 μmol/L), followed by 50 μL of formic acid solution (10%, v/v), were added to an aliquot (100 μL) of serum or deionized water. After vortexing the sample, 500 μL of ethyl acetate was added for extraction. The sample was mixed well with the extractant and centrifuged at 2750× g for 15 min at 4 °C. Next, an aliquot (400 μL) of organic extract was transferred to the test tube. For the second extraction cycle, 500 μL of ethyl acetate was added to the sample again. After mixing and centrifugation, 400 μL of organic extract was transferred to the same test tube with the first aliquot of extract. Combined extracts were evaporated under the stream of nitrogen (40 L/min) at 40 °C. The dry residue was dissolved in 400 μL of the mobile phase solution (0.2% acetic acid and 5% acetonitrile in water, v/v), mixed well and transferred to a test plate for further UPLC-MS/MS analysis.