For the derivatization process, 200 μL of the extract was transferred
to polypropylene centrifuge tubes, and 400 μL of saturated
sodium bicarbonate solution and 800 μL of dansyl chloride solution
were added. Next, the tubes were agitated by vortex for approximately
30 s and kept in the dark in a hot water bath at 60 °C for
5 min. After this step, 200 μL of L-proline solution was added, and
the tubes were again agitated by vortexing for 30 s; the agitated
solution was kept in the dark at room temperature for 30 min. After
this time, 1000 μL of toluene was added to the solution, which was
agitated for 1 min followed by centrifugation at 4350g for 10 min at
4 °C to separate the phases. The organic phase (supernatant) was
recovered using an automatic pipette into a 5-mL test tube that was
transferred to the sample concentrator for evaporation by adding
nitrogen flow for 10 min at 60 °C.
The obtained extract was dissolved in 600 μL of acetonitrile and
filtered in a filter unit with polytetrafluoroethylene (PTFE) membrane
(pore size of 0.45 μm, diameter from 13 to 15 mm; Millipore
Corp, Milford, MA, USA), and the filtrate was reserved for injection.