Endocrine Abstracts (2010) 21 P339

Analysis of cortisol by stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS): pitfalls of rapid LC-MS/MS analysis of clinical samples

Natalie Homer1, Scott Denham1, Roland Stimson1, David Watson2, Brian Walker1 & Ruth Andrew1

1University of Edinburgh, Edinburgh, UK; 2University of Strathclyde, Glasgow, UK.

The glucocorticoid hormone, cortisol, regulates fuel metabolism, inflammation and stress–responses. Its circulating concentrations are tightly controlled by the hypothalamic–pituitary–adrenal axis. However, 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) generates additional cortisol in tissues, by reduction of inert cortisone. Using a tracer (9,11,12,12[2H]4-cortisol; d4-cortisol), the velocity of 11βHSD1 can be determined as the rate of appearance of 9,12,12[2H]3-cortisol (d3-cortisol), generated via the intermediate, 9,12,12[2H]3-cortisone (d3-cortisone).

Historically, quantitation of cortisol has been performed by gas-chromatography–MS, achieving limits of detection (LODs) of ~100 ng/ml, in biological fluids, following derivatisation and with run times of 45 min. Increasingly LC–MS/MS assays are being developed, with improved selectivity and more rapid throughput.

To quantify cortisol, cortisone and their isotopomers, an LC–MS/MS assay was developed. Plasma steroids (extracted in chloroform) were resolved in 11.5 min on an Allure Biphenyl column (50×2.1 mm; 3.5 μm) prior to MS detection. The protonated analytes gave the mass transitions; cortisol (m/z363→121), d3-cortisol (m/z366→121), d4-cortisol (m/z367→121), cortisone (m/z361→163) and d3-cortisone (m/z364→164). The assay had superior LOD (2 ng/ml), compared with GCMS, with inter-assay precisions (5.5, 8.6%) and accuracies (−4.5, −3.8%) for cortisol and cortisone respectively.

While this assay achieved improved performance and throughput, unexpectedly a further substance co-eluted with d3-cortisone plasma extracts, yielding an ion undergoing the same mass transition. To resolve d3-cortisone from the interferent, prolonged chromatography (30 min), on a longer column, was necessary. Further investigation revealed that the precursor ion was the mass+2 isotopomer of an ion with m/z362. The accurate mass (362.11703 amu), determined by Fourier Transform-MS, identified the interfering substanceent as omeprazole sulphone. Three of the four participants from whom samples contained this interferent recalled recently taking recent omeprazole.

Thus LC–MS/MS as an excellent tool to enhance throughput and specificity of analysis of glucocortiocids. However care is required in screening of pre-existing medication in clinical studies, before executing rapid chromatography with biological samples.

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