Ascentis express hilic column

Quantification of glutamate was performed by liquid-chromatography coupled to mass spectrometry (LC-MS/MS). Chromatographic separation was performed on an Agilent 1200 series (Waldbronn, Germany) using an Ascentis Express HILIC column, 50×2.1 mm with 2.7 Rm particle size from Supelco (Belfonte, PA) maintained at 25°C throughout the analysis, a mobile phase acetonitrile and water (50 mM ammonium acetate) with a flow rate of 0.6 mL min-1. The volume injected was 10 RL. The mobile phase involved a gradient starting at 87% of ACN which was maintained for 3 minutes. Then, from min 3 to 10 the ACN content was decreased to 20% and increased again to 87% at min 12.5. Glutamate was eluted at 6.5 minutes. The mass detection system was an Agilent 6410 Triple Quad (Santa Clara, CA) using positive electrospray ionization with a gas temperature of 350°C, gas flow rate of 12 L min-1, nebulizer pressure of 45 psi, capillary voltage of 3500 V, fragmentor of 135 V and collision energy of 10 V.

For the determination of the intracellular content of arginine, cells were rapidly washed with PBS and the intracellular pool, extracted with a 10 min-incubation in acetonitrile/water (1:1) at 4°C, was analyzed by HPLC-ESI-MS/MS as previously described (27 (link)), with minor modifications. Briefly, HPLC separation was carried out on Ascentis Express HILIC column (Supelco, Bellefonte, PA, USA) at 35°C. Analytes (injection volume corresponding to 10 µl) were chromatographically separated under optimized gradient; mass spectrometric analyses were carried out using an AB SCIEX 4500 Q-TRAP (AB Sciex, Foster City, CA, USA) with a Turbo Ion Spray probe in positive mode. The monitored transitions for l-arginine and ¹⁵N₂-Arginine (used as internal standard) were 174.739 m/z→70.031 m/z and 176.739 m/z→70.031 m/z, respectively. Protein content in each condition was determined using a modified Lowry procedure (28 (link)) and l-arginine content was expressed as nmol/mg of protein.