Agilent lc ms ms 1200s 6410

A volume of 200 µL of enzyme extraction was filtered and 20 µL was injected in a rheodyne valve into an HPLC–ESI–MS/MS system (Agilent LC–MS/MS 1200s-6410, Agilent Technologies, Santa Clara, CA, USA) equipped with an Astec Chirobiotic™ column (150 × 21 mm, 5 µm pore size), the mobile phase consisted of a mixture of 0.1% of formic acid (A) and acetonitrile (B) 95.5:0.5 respectively, the flow rate was 0.5 mL/min. The detector was set in MRM mode (multiple reaction monitoring) at 4000 V, 35 psi, and a 9 mL/min flow rate of nitrogen. Proline (116 → 70 m/z), Gly-betaine (235 → 118 m/z) and were used as standards (Sigma-Aldrich, MO, USA).

Fresh tissue (100 mg) was ground to a fine powder with liquid nitrogen and extracted in 1 mL of methanol: formic acid: water 15:4:1. The mixture was centrifuged at 10,000 rpm for 10 min at 4 °C. The supernatant was recovered and filtered (0.22 µm). A volume of 20 µL of the extract was injected in a rheodyne valve into an HPLC–ESI–MS/MS system (Agilent LC–MS/MS 1200s-6410, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18-reversed-phase column (150 × 4.6 mm, 5 µm, XDB-C18, Agilent Technologies, Santa Clara, USA), the mobile phase consists in a solution of 0.1% formic acid, the running was set at a flow rate of 0.3 mL/min at room temperature. The detector was set in MRM mode (multiple reaction monitoring) at − 4500 V, 25 psi, and a 10 mL/min flow rate of nitrogen. ABA (263 → 153 m/z) was used as standard (Sigma-Aldrich, MO, USA) and d6-ABA (269 → 159, Olchemim Ltd., Czech Republic) as an internal standard.