Milliq grade deionized water

To stabilize ascorbate and to precipitate proteins, 200-µL aliquots of freshly prepared or partially thawed plasma were mixed with 200 µL of precooled 10% (w/v) meta-phosphoric acid (MPA, Sigma-Aldrich, Munich, Germany) containing uracil (50 µM, Sigma-Aldrich) as an internal standard. The samples were kept on ice for 40 min and then diluted with 200 µL of MilliQ-grade deionized water (Merck Millipore), vortexed and centrifuged at 25,155×g for 15 min at 4 °C. The supernatants (200 µL) were purified by ultrafiltration using AcroPrep Advance 96-Well Filter Plates 10 K (Pall), and injected into Waters Acquity ultra-performance liquid chromatographic (UPLC) system. The method was validated with the reference material from Chromsystems.

To precipitate proteins, 200-µL aliquots of freshly thawed plasma were mixed with 200 µL of anhydrous ethanol (POCH) containing an internal standard (0.5 mL IS/10 mL EtOH) (Vitamins A and E in Serum/Plasma—HPLC, Chromsystems), vortexed and left for 10–15 min. Then, 400 µL MilliQ-grade deionized water (Merck Millipore) and 800 µL of hexane (Aldrich) were added to extract the vitamin. The samples were shaken vigorously for 2 h and centrifuged at 25,155×g for 10 min. Then, 400 µL of the upper layer (hexane) were collected, dried in Speed-Vac system (8 min), and dissolved in 100 µL of mobile phase (acetonitrile and methanol (Sigma-Aldrich), 80:20 (v/v), HPLC-grade). The samples were shaken vigorously overnight, centrifuged at 25,155×g for 10 min, and supernatants were injected into HPLC system. The method was validated with the reference material from Chromsystems.