Gas chromatographic-mass spectrometric method for measuring stable isotope enrichments of underivatized cortisol from...

Isotope dilution methods utilizing quantification of stable enrichment by mass spectrometry has been used recently to determine cortisol production rates in humans. Studies of steroid production and utilization rates require frequent blood sampling, and while stable isotopes are safe for pediatric and perinatal use, the required blood volumes may be problematic using published methodologies. We have developed methods to isolate and quantify the plasma enrichment of cortisol with its stable isotope, [9,12,12-2H3]cortisol using a simple four-step isolation procedure and gas chromatography-mass spectrometry. Isolation is semi-quantitative, but reproducible (mean recovery; 51.5 +/- 5.4% coefficient of variation) and the method does not require derivation. In human studies, this method can determine plasma enrichments between 1 and 10 mol% at plasma concentrations of 2 micrograms/dl or more; only 1 ml of plasma is required. The concentrations of labelled cortisol added to plasma are low (0.2-0.5 micrograms/dl) and are not expected to interfere with the sensitive hypothalamic-pituitary-adrenal feedback system. We conclude that reproducible quantification of stable cortisol isotope enrichment can be achieved from small plasma volumes.