Canrenone and mifepristone (RU486) are respectively mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) antagonists commonly used to block or displace steroid for clinical pharmacological or research purposes. The aim of this study was to develop and validate a sensitive, quantitative assay for the combined analysis of glucocorticoids (cortisol, cortisone, corticosterone, and 11-dehydrocorticosterone), mineralocorticoids (aldosterone), canrenoate, and mifepristone.
HPLC mass spectrometric method development was carried out on an ABI 5500 QTrap triple quadrupole mass spectrometer with an Acquity UPLC System. Under mixed mode electrospray ionisation and collisional activation, the following parentproduct transitions were monitored; m/z cortisol: 363→121; cortisone: 361→77; corticosterone: 347→91; 11-dehydrocorticosterone: 345→121; aldosterone: 359→331; canrenone: 341→91, and RU486: 430→134. Ions for corresponding deuterated internal standards were incorporated. Analytes eluted between 2 and 6 min. Optimal separation was achieved using water (0.1% formic acid)/acetonitrile (0.1% formic acid) at 70:30, 0.5 ml/min on a Waters Sunfire C18 HPLC column (3.5 μm; 150×2.1 mm) at 30 °C. A gradient rising to 90% acetonitrile was applied, with a total run time of 9 min.
Satisfactory recoveries of all analytes (mean 83.56% (relative S.D. 11.6%)) were achieved following liquidliquid extraction of 100 μl plasma with chloroform (1:10). Intra-assay validation parameters for quantitative analysis were assessed with three replicates at low, medium, and high concentration. The limit of quantitation was ~0.1 ng for endogenous steroids and ~5 ng for the drugs with mean precision of 9.4%. The assay was linear (r2~1) over a range of concentrations (0.1100 ng).
This assay is suitable to detect expected endogenous steroid levels and drug doses in one aliquot of ~200 μl of plasma and will allow rapid simultaneous profiling of circulating adrenal hormones.