Serum Metabolite Profiling by LC-MS/MS

Blood samples were collected from the participants before the second HD session of the week and centrifuged obtaining serum samples and stored at −80 °C until analysis. Sixty microliters of serum were treated by 60 µL of deproteinizing solution. The mixture was mixed for 30 s and centrifuged at 14.000× g rpm for 15 min. 10 µL of clean supernatant was injected into the chromatographic system. Compounds were detected using a LC–MS/MS analytical method [16 (link),17 (link)]. Chromatographic separation of analytes was made utilizing an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, Santa Clara, CA, USA), on a F5 column (Phenomenex, Torrance, CA, USA) equipped with a security guard pre-column (Phenomenex, Torrance, CA, USA). The mobile phase included a solution of 0.1% aqueous formic acid and 100% acetonitrile; elution was realized at a flow rate of 300 μL/min, using an elution gradient, as previously described [17 (link)]. The mass spectrometry was realized on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA) supplied with a Turbo Ion Spray source, and the detector was set in the positive ion mode, as previously described [17 (link)]. The instrument was set in the Multiple Reaction Monitoring (MRM) mode. Data were collected and analyzed by the Analyst 1.5.1 Software.