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AS103: Automating the Extraction and Analysis of Urinary Free Cortisol Using Instrument Top Sample Prep (ITSP), GERSTEL MultiPurpose Sampler and Agilent LC-MS/MS

Martin Perkins

13th September 2019


Automating the Extraction and Analysis of Urinary Free Cortisol Using Instrument Top Sample Prep (ITSP), GERSTEL MultiPurpose Sampler and Agilent LC-MS/MS

The measurement of urinary free cortisol (UFC) is used in the investigation of possible Cushing’s syndrome. Cortisol is a steroid-hormone synthesized from cholesterol by a multienzyme cascade in the adrenal glands. It is the main glucocorticoid in humans and acts as a gene-transcription factor influencing a multitude of cellular responses in virtually all tissues.

Only a small percentage of circulating cortisol is biologically active (free), with the majority of cortisol inactive (protein bound). As plasma cortisol values increase, free cortisol (i.e., unconjugated cortisol and hydrocortisone) increases and is filtered through the glomerulus.

UFC in the urine correlates well with the concentration of plasma free cortisol. UFC represents excretion of the circulating, biologically active, free cortisol.

Cushing’s syndrome is a hormonal disorder caused by prolonged exposure of the body’s tissues to high levels of the hormone cortisol. Cushing’s syndrome is relatively rare and most commonly affects adults aged 20 to 50. Signs and symptoms of Cushing’s syndrome vary, but most people with the disorder have upper body obesity, a rounded face, increased fat around the neck, and relatively slender arms and legs. Children tend to be obese with slowed growth rates.

Presented in this application note is a fully automated miniaturised solid phase extraction (SPE) method for the extraction and clean-up of UFC, followed by tandem mass spectrometry detection. Performance data is derived from spiked urine and commercially available QC materials. Currently the automated extraction takes four and a half minutes, which fits well within the LC cycle time of seven minutes, this allows for highest possible throughput for this clinically important assay.

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