Phenylalanine d 5

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 Then, 100 µL of supernatant was withdrawn and 200 µL of HCOOH 0.1% v/v in H₂O was added and the solution was vortexed 10 s and centrifuged at 10000 × g (4 °C, 10 min).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.

Reverse-osmosed ultrapure water was obtained from a Milli-Q Plus185 system (Millipore, Billerica, MA, USA). A MS grade methanol (MeOH), used for standards preparation, and acetonitrile (ACN) were obtained from Fisher Scientific (Hampton, NH, USA). Analytical grade ammonia solution (28%, GPR RECTAPUR^®) and acetic acid glacial (AnalaR^® NORMAPUR^®) were obtained from VWR Chemicals (Radnor, PA, USA).
Arachidonic acid, LPC 14:0, LPC 16:0, LPC 17:0, LPC 17:1, LPC 18:0, LPC 18:1, LPC 19:0, LPE 18:0, LPI 16:0, LPI 20:4, palmitoleic acid, oleamide, SPA-1P, sphinganine-C17, sphingosine, S1P, sphingosine d7, LPC 18:1 d7 were purchased from Avanti lipids (Birmingham, AL, USA). Bilirubin, lactic acid, lauric acid, oleic acid, adenosine, arginine, betaine, carnitine, cortisol, creatine, creatinine, hexanoylcarnitine, hippuric acid, hypoxanthine, leucine/isoleucine, phenylalanine, proline, propionylcarnitine, urea, carnitine d3, isoleucine d7, and palmitic acid d31 were purchased from Sigma-Aldrich (Darmstadt, Germany). phenylalanine d5 and valine d8 were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA).