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Sample pre-treatment
Dilute 150 uL of human plasma (spiked with 25 ng/mL and 125 ng/mL of cortisone and prednisolone respectively) with 150 uL of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted. Mix briefly (3-5 sec).
Sample loading:
Load the sample from pre-treatment step above onto the Novum plate and apply a short and gentle pulse of vacuum (~ 10" of Hg for 20 secs) until the sample has completely entered the media.
Wait for 5 minutes.
Elution:
Dispense 1000 uL of ethyl acetate onto the SLE media and allow the solvent to elute by gravity (~ 5 min elution time) and collect the eluant.
Apply vacuum at 5" of Hg for 45 secs to complete the extraction.
NOTE: Prolonged application of vacuum will result in elution of plasma out of the SLE media and into the final extracted solvent.
Dry down:
Evaporate the final extract to complete dryness under slow stream of N2 at 40°C
Reconstitute:
Reconstitute the dry residue in 150 uL of initial mobile phase fortified with cortisol-D4.