4.3. Pre-analysis preparation of the HPLC

1. Prepare the mobile phase (1l) as follow:

   1.1.  700 ml polished water (this is ddH20 from which all impurities were removed)
   1.2.  150 ml methanol
   1.3.  150 ml acetonitrile
   1.4.  0.433 g sodium dodecyl sulphate (SDS)
   1.5.  10.349 g sodium phosphate monobasic
   1.6.  1.47g sodium citrate
   1.7.  Adjust pH to 5.6 using phosphoric acid, delivered a drop at a time while stirring.

2. Degas the mobile phase for the HPLC in an ultrasonic bath for 5 min prior to running any samples to prevent air from entering the column and detector cells.

3. Set the mobile phase flow rate to 1.0 ml/min (normal pressure ~95 bar; set max pressure to 140 bar).

4. Apply current to the ECD cells (e.g. Channel 1, 650mV; Channel 2, 425mV; Channel 3, 175mV; Channel 4, -175V).

The amount of current per channel will need to be adjusted to optimize peak responsiveness as per the manufacturers recommendations; it will take approximately 45 min for channel voltage to equilibrate.

5. During the equilibration process, prepare the external (ES) and internal standards (IS).