Reprosil pur 120 c18 aq analytical column

Shotgun proteomics analyses were performed using an EASY-nLC^TM 1200 UHPLC system (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific) operating in the data-dependent acquisition (DDA) mode. A sample volume corresponding to 2 μg of total peptides reconstituted in 0.1% FA was injected onto an Acclaim PepMap100 C18 Nano-Trap column (2 cm × 100 μm, 5 μm; Dionex, Sunnyvale, USA). Peptides were separated on a Reprosil-Pur 120 C18-AQ analytical column (15 cm × 150 μm, 1.9 μm; Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) using a 75 min linear gradient from 5 to 100% eluent B (0.1% FA in 80% acetonitrile) in eluent A (0.1% FA in H₂O) at a flow rate of 600 nl/min. For DDA, the Orbitrap Fusion Lumos mass spectrometer was operated in positive polarity mode with spray voltage of 2.3 kV and capillary temperature of 320 °C. Full mass spectrometry (MS) scans from 300 to 1500 m/z were acquired at a resolution of 60,000 resolving power (at 200 m/z) with an AGC target value of 4 × 105 and a maximum ion injection time of 50 ms. The MS2 scans were acquired at a resolution of 15,000 resolving power (at 200 m/z) with an automatic gain control (AGC) target value of 5 × 104, a maximum ion injection time of 35 ms, and a normalized collision energy of 36%.

Ascorbic acid in 25 mg of freeze-dried samples were extracted with a mixture of 550 µL of freshly prepared 10% (w/w) meta-phosphoric acid (MPA) and 50 µL of 20% (w/w) tris-(2-carboxyethyl)-phosphine (TCEP, C₉H₁₅O₆P·HCl) solution as the reduction agent; samples were incubated for 5 min on ice (in the dark, protected from light), then vortex-mixed for 3 min, and centrifuged at 17,000 × g for 10 min; 20 µL of supernatants were analysed on AA content using a Shimadzu Prominence HPLC, a Reprosil-Pur 120 C18 AQ analytical column (5 µm, 250 × 4.6 mm, Dr. Maisch GmbH, Ammerbuch, Germany), sodium dihydrogen-phosphate buffer (0.1 M NaH₂PO₄ x 3H₂O, set to pH 2.5) as a mobile phase at a flow rate of 1 mL/min and an UV-Vis detector (SPD-20A, Shimadzu, Kyoto, Japan) set at 245 nm [16 (link)]. TCEP, MPA, and reagents were from Carl Roth GmbH + Co. KG, Karlsruhe, Germany. The recommended nutrient intake (RNI) was calculated based on 45 mg/day recommendation of Vitamin C for women 19–50 years [15 ].