Abstract
Existing methods for cortisol analysis are very diverse. This study focuses on developing an optimized sample preparation procedure for cortisol from plasma. The evaluation resulted in a rapid, clean, and reproducible method using Novum Simplified Liquid Extraction (SLE) 96-well plates.
Introduction
Cortisol or hydrocortisone is a corticosteroid secreted by the adrenal cortex. Cortisol is synthesized from cholesterol and may be found in the blood bound to globulin or in free-form. Cortisol has an anti-inflammatory effect and aids in carbohydrate metabolism, renal function, and the promotion of gluconeogenesis. Measurement of plasma cortisol levels is useful when studying the functions of the adrenal cortex, including Cushing’s syndrome (hypercortisolism), Addison’s disease (hypocortisolism), and adrenal tumors. Abnormal cortisol levels may be linked to prostate cancer, depression, and schizophrenia. The aim of this work is to introduce a fast and effective plasma cortisol extraction procedure using Novum SLE which results in a clean extract with high recovery. To determine the most effective procedure, two different extraction solvents were examined; dichloromethane (methylene chloride or DCM) and a more polar elution solvent, methyl tert-butyl ether (MTBE)/ethyl acetate (EtOAc) (50:50).
Experimental Conditions
Extraction Procedure
Sample Pre-treatment
- Dilute 200 μL of human plasma (spiked with cortisone at 25 ng/mL) with 200 μL of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted. Vortex briefly (3-5 sec).
Sample Loading
- Load the sample from the pre-treatment step above onto the Novum SLE MAX plate (Part No.8E-S138-5GA) and apply a short and gentle pulse of vacuum (~ 5” of Hg for 5-10 sec) or until the sample has completely entered the media.
- Wait for 5 minutes.
Elution
- Dispense 900 μL of elution solvent onto the Novum SLE media and allow the solvent to elute by gravity (~ 5 min elution time) and collect the eluant.
- Repeat with another 900 μL of elution solvent and collect the eluant.
- Apply vacuum at 5” of Hg for 20-30 secs to complete the extraction.
NOTE: Prolonged application of vacuum will result in elution of plasma from the Novum SLE media and into the final extracted solvent.
Dry Down
- Evaporate the final extract to complete dryness under a slow stream of N2 at 40 °C.
- Reconstitute the dry residue in 200 μL of initial mobile phase fortified with cortisol-D4.
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