Relative quantification of GSH by LC-MS/MS
Relative quantification of GSH was achieved on an Agilent 1200 series HPLC system (Agilent Technologies) coupled to a tandem mass spectrometer API 3200 (Applied Biosystems, Darmstadt, Germany) via electrospray ionization (ESI) in positive ionization mode. An aliquot of the raw extract from GSL analysis (see above) was injected. A Zorbax Eclipse XDB-C18 column (Agilent Technologies) was used for separation. 0.05% formic acid and acetonitrile were used as solvent A and B, respectively, at a flow rate of 1.1 mL/min with the following profile: 0 - 0.5 min, 3 - 15% B; 0.5 - 2.5 min, 15% - 85% B; 2.5 - 2.6 min, 85 - 100% B; 2.6 - 3.5 min, 100% B, 3.5 - 3.6 min, 100% B - 3% B and 3.6 - 6.0 min 3% B. The MS parameters were optimized as follows: ion spray voltage, 5500 V; turbo gas temperature, 650°C; collision gas, 3 psi; curtain gas, 35 psi; ion source gas 1, 60 psi; ion source gas 2, 60 psi. MRM for the parent ion - product ion was set as follows: m/z 308.1 - 179.1 (CE, 17 V; DP, 46 V) for GSH. Relative quantification was accomplished and expressed in relative peak area units of the LC-MS/MS signal per mg fresh weight.