Analytical Standards Identification Protocol

Individual stock solutions of 15 analytical standards from different chemical classes were prepared by adding 1 mg of each of the following analytical standard into 1 mL of water (or other solvent, if specified): 1-methyluric acid (water + 5 µL sodium hydroxide 5 mM), 3-methylhistidine, ADMA, caffeine, CDP-choline, creatinine, dAMP, glutaric acid, glycero-phosphocholine, methionine, phenylpyruvic acid (ethanol), serine, sphingosine (methanol), taurine and threonic acid. Next, 200 µL of each standard solution were pipetted into a 5 mL Eppendorf tube, together with 40 µL of formic acid and 960 µL of ACN, resulting in 4 mL of a standard mix solution at 50 ppm. For data acquisition, 5 µL of standard mix solution was injected into a LC-MS system equipped with an Agilent 1290 UHPLC device (Agilent Technologies, Santa Clara, CA, USA) coupled with a SCIEX 5600 QTOF (AB Sciex LLC, Framingham, MA, USA). An Acquity UPLC BEH Amide Column (130 Å, 1.7 µm, 2.1 mm × 100 mm) was used in association with the respective pre-column. An IDA experiment (information dependent analysis, also known as data dependent analysis or DDA) was performed using a 50–1000 m/z range, with a 250 ms accumulation time for MS¹ data. MS² data were acquired using the same m/z range and a 1000 cps threshold. In addition, 50 mDa was used as mass tolerance with a maximum number of candidate ions per cycle set at 20. Dynamic background subtract and dynamic accumulation were also employed, with an accumulation time of 100 ms and collision energy set at 20 V.