Luna hilic

All exerkines were determined using plasma samples containing EDTA except for meteorin-like, that was determine in plasma samples containing heparin. BAIBA was determined by high-performance liquid chromatography (HPLC), according to the previously described methodology (Molfino et al., 2017) , by Agilent Liquid Chromatography System series 1100 (Agilent Technologies, USA) using columns from Phenomenex Luna HILIC (100 × 30 mm, Phenomenex, CA, USA). The kits used for the determination of the other exerkines plasma levels are pointed in Supplementary Table 1.

The meconium and calibration samples were analyzed on an Acquity UPLC H-Class from Waters (Milford, MA, USA). Chromatographic development was accomplished with six columns: Ascentis Express C18, 150 × 2.1 mm, 2.7 µm from Supelco (Bellefonte, USA); Ascentis Express RP-Amide, 150 × 2.1 mm, 2.7 µm from Supelco (Bellefonte, USA); Ascentis Express Phenyl-Hexyl, 150 × 2.1 mm, 2.7 µm from Supelco (Bellefonte, USA); Acclaim Trinity P1, 150 × 2.1 mm, 3 µm from Thermo Fisher Scientific (Asheville, NC, USA); Ascentis Express HILIC, 150 × 2.1 mm, 2.7 µm from Supelco (Bellefonte, USA); and LUNA HILIC, 150 × 2.0 mm, 3 µm from Phenomenex (Torrance, CA, USA).

The liquid chromatographic system was an Agilent 1260 Infinity High Pressure LC (HPLC) fitted with a degasser, a binary high-pressure gradient pump, a thermostated column compartment and an autosampler module. Chromatographic separation was achieved using a Phenomenex Luna HILIC (hydrophilic interaction liquid chromatography) 200 Å (150 x 3 mm, 5 µm) column, with a mobile phase composed of A) 5 mM AmOAc in ultrapure water and B) MeCN, at a flow rate of 0.4 mL/min. The gradient was as follows: 0-0.5 min: 95% B; 0.5-5 min: 95%-90% B; 5-12.5 min: 90%-70% B; 12.6-14.6 min 30% B; 14.7 min 95% B.
The total run time including column equilibration was 19 min. The injection volume was optimized based on peak shape and set to 2 µL. The LC system was coupled to an Agilent 6410 triple quadrupole mass spectrometer with an electrospray interface (ESI) for the detection and quantification of compounds. Source parameters were as follows: gas temperature 350 °C, gas flow 10 L/min, nebulizer 40 psi, capillary voltage 4000 V. The mass spectrometer compound dependent parameters were optimized for each compound individually (Table 1).