Thawed plasma samples (0.4 mL) were spiked with 20 µL of 500 ng/mL of EC-d5 in water, gently mixed, and incubated at 4°C for 10 min. Then 40 µL of 20% (w/v) 5-sulfosalcylic acid in water was added to induce protein precipitation and samples were vigorously mixed for 10 s, followed by 20 min of incubation at 4°C. The precipitated proteins were centrifuged at 17,000 g and 4°C for 15 min. Supernatant (0.35 mL) was recovered and washed with 0.3 mL of hexane to remove neutral lipids. After removal of hexane, the aqueous plasma extract was added with 120 mg of NaCl, and then 1.6 mL of ethyl acetate. Samples were vigorously mixed for 30 s and then centrifuged at 2000 g and 4°C for 10 min. Then upper organic layer was recovered, and the solvent was removed under a stream of nitrogen until about 60 µL remained.