Sample Preparation

• Step 1
  Pipette 300 µL of plasma sample (calibrator or control) into a glass centrifuge tube then add 20 µL of internal standard solution and vortex for 5 sec.
• Step 2
  Add 600 µL of Reagent-1 and vortex for 15 sec. then, add 1 ml of Reagent-2. After agitating for 3 min. at room temperature, centrifuge at 4000 rpm for 2 min.
• Step 3
  Discard 700 µL of the upper phase and add Reagent-3 (solid) to the tube before vortexing 30 sec.
• Step 4
  Add 200 µL of Reagent-4 and 2 ml of Reagent-5 into the tube.
• Step 5
  Shake the tube for 7 min. at room temperature after that, centrifuge at 4000 rpm for 3 min.
• Step 6
  Evaporate 1.6 ml of upper phase to dryness.
• Step 7
  Reconstitute using 100 µL of Reagent-1 and transfer to the inserted HPLC vial prior to injection.