2.3.3. Determination of L-Ascorbic Acid

The determination of L-ascorbic acid was conducted as described by Iwase et al. [15].

A stock solution 10 mmol L⁻¹ L of ascorbic acid was prepared in water. The stock solution was further diluted with water up to 40 µmol L⁻¹.

Immediately after blood collection and centrifugation, 200 µL of plasma were deproteinized with 400 µL of meta-phosphoric acid (10% wt/wt), vortexed for 15 s, and centrifuged at 10,800 rpm for 7 min. Following this step, 350 µL of supernatant were directly analyzed or kept in vials at −80 °C until analysis.

L-ascorbic acid content was determined using a reversed-phase column (Zorbax SB C18 Stable Bond 4.6 × 250 mm, 5 µm; Agilent, Santa Clara, USA) at 27 °C. KH₂PO₄ 20 mmol L⁻¹ and 0.2 mmol L⁻¹ of EDTA solution (pH = 3.0 adjusted with ortho-phosphoric acid solution) was used as the mobile phase. The flow rate was set at 1 mL min⁻¹ and the wavelength used for the photometric detection was 244 nm. Sample injection volume was 20 µL and the run time was 14 min.