Measurement of plasma thiols
SEPARATION
Amino acids, total glutathione (tGSH), total cysteine (tCys) and total homocysteine (tHcy) were measured at all six time points using plasma samples from blood collection. Plasma were stored at −80 °C. After thawing and vortexing, 10 µL of plasma, calibration standards, QC samples, and blank matrix (water) were added to 96 well plates. Internal standard working solution (10 µL) and 10 μL dithioerythritol (DTE) [Chromatographic separation was performed on Phenomenex Kinetex Core Shell C18 (100 × 4.6 mm, 2.6 μm) and temperature regulated at 30 °C. The mobile phases were (A) water with 0.5% formic acid, 0.3% heptafluorobutyric acid (HFBA) and (B) acetonitrile at a flow rate of 0.8 ml/min. The separation was achieved with a linear gradient from 100% (A) to 80% (A) over 6 min and 100% (A) to 40% (A) over 10.5 min, followed by a linear gradient back to 100% (A) over 12 min. The injection volume was 15 µL. Then each amino acid was identified by MS, corresponding to each particular internal standard. The concentration of each amino acid was determined from the ratio of analyte peak area / internal standard peak area against a linear multiple point calibration curve. For calibration and quality assurance, we used NIST standard reference material, ensuring alignment with established standards and facilitating result accuracy and comparability. In the case of amino acids, we compared internal quality controls against Erndim reference controls, further validating the accuracy and reliability of our measurements. [