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