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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).
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.
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.
Evaporate the final extract to complete dryness under slow stream of N2 at 40°C
Reconstitute the dry residue in 150 uL of initial mobile phase fortified with cortisol-D4.
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