Caffeine d9

Urine samples, once collected were kept at −80 °C until analysis. Samples were thaw at room temperature on ice, vortexed for 10 s and centrifuged at 10000 × g (4 °C, 10 min). Then, 100 µL of supernatant was withdrawn and 200 µL of HCOOH 0.1% v/v in H₂O was added and the solution was vortexed 10 s and centrifuged at 10000 × g (4 °C, 10 min). No molecular weight cutoff (MWCO) filters were used during sample preparation. Thereafter, 100 µL of the supernatant was transferred to a 96 well plates where each sample was spiked with 5 µL of an internal standard solution containing Phenylalanine-D₅ (Cambridge Isotopes Laboratory Inc., Andover, MA, USA), caffeine-D₉ (Toronto Research Chemicals, Toronto, Ontario, Canada), leukine enkephalin (Sigma-Aldrich Química SA, Madrid, Spain) and reserpine (Sigma-Aldrich Química) in H₂O:CH₃OH (1:1, 0.1% v/v HCOOH), at a final concentration of 1 µM each. Control blanks were prepared by replacing urine by H₂O. A quality control (QC) sample was prepared by mixing 5 µL of each prepared sample. All solvents were of LC-MS grade and were purchased from Scharlau (Barcelona, Spain). Ultra-pure water was generated with a Milli-Q water purification system (Merck Millipore, Darmstadt, Germany). Formic acid (≥95%) was obtained from Sigma-Aldrich Química.