Plasma Preparation and Extraction for UPLC-MS

Plasma samples were collected in vacuum tubes using EDTA as an anticoagulant (EDTAK3, 3 mL), mixed gently by inverting 8–10 times, and kept at 2–8 °C until centrifugation. Blood samples were centrifuged at 1200× g for 15 min within 30 min after blood collection. Plasma was separated from the red blood cells as soon as possible (maximum, 10–15 min) after centrifugation and kept at −80 °C until analysis. 30 μL of plasma sample was mixed with 30 μL of internal standard 0.5 μM in 10% ammonium hydroxide (UPLC grade) solution and vortexed for 15 s. 60 μL of 200 nM ZnSO₄ (+5 °C) was added and vortexed for 15 s. 30 μL of methanol (+5 °C) (UPLC grade) was added and vortexed for 15 s. The mixture was centrifuged for 10 min at 2841× g at room temperature. 30 μL of the supernatant was mixed with 30 μL of formic acid 5% in LC-MS Certified Clear Glass 12 × 32 mm vials (Waters, Manchester, UK, product no. 186005662CV). Vials were transferred to an autosampler (set to 5 °C) that injected 1.5 μL into the ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) system [36 (link)].