Introduction
Corticosteroids exhibit anti-inflammatory properties and are widely abused within the sports industry. Hence there is a need for more sensitive analytical tools to detect and confirm these classes of drugs. Chromatographic separation is essential for detection and confirmation of corticosteroids because cortisone and prednisolone are isomeric compounds. A simple automated extraction method using a Tecan Freedom EVO® 100 liquid handler and Phenomenex’s Novum Simplified Liquid Extraction (SLE) 96-well plate is employed to extract corticosteroids from plasma samples. A Kinetex 2.6 μm, 50 x 3 mm core-shell Biphenyl HPLC/UHPLC column is used to successfully separate these two compounds which will be beneficial for detection and confirmation of the two isomers.
Experimental Conditions
Extraction Procedure
Sample pre-treatment
- Dilute 150 μL of human plasma (spiked with 25 ng/mL and 125 ng/mL of cortisone and prednisolone respectively) with 150 μL 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 SLE MINI plate (Part No. 8E-S138-FGA) and apply a short and gentle pulse of vacuum (~5-10” of Hg for 20 secs) until the sample has completely entered the media.
- Wait for 5 minutes.
Elution
- Dispense 1000 μL of ethyl acetate onto the Novum 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.
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