Endocrine Abstracts

Canrenone is a mineralocorticoid receptor antagonist used as a diuretic agent to treat hypertension. It is the major active metabolite of spironolactone and may be quantified in clinical studies either to ensure compliance or to gain information about pharmacokinetic-pharmacodynamic interactions.

The aim of this study was to develop and validate a sensitive, quantitative assay for the analysis of canrenone in plasma.

HPLC mass spectrometric method development was carried out on a TQ4 Quantum Discovery triple quadrupole mass spectrometer with an Aria CTC HPLC autosampler system. Under positive electrospray ionisation the major ion detected was the protonated molecular ion (M+H)⁺m/z 341 for canrenone. Alfaxalone (precursor ion m/z 333; 1 μg) was used as the internal standard. Under collisional activation, the major fragmentation ions were m/z 107.1 and 91.1 for canrenone and m/z 297.3 and 315.3 for alfaxalone. The fragmentation ions were used as quantifier and qualifier ions respectively to add specificity to the assay. Optimal separation of the steroids was achieved using ammonium acetate (5 mM)/methanol at (60:40, 0.3 ml/min) on a Waters C18 T3 Atlantis HPLC column (3 μm; 100×2.1 mm) at 25 °C. A gradient rising from 40 to 90% methanol was applied, with a total run time of 8 minutes.

Validation of quantitative parameters was performed using six intra- and inter-assay replicates. Satisfactory recoveries of canrenone and alfaxalone (106.2% (relative standard deviation (RSD) 9.5%) and 102.2% (RSD 4.7%)) were achieved following liquid-liquid extraction of only 25 μl plasma with ethyl acetate (1:10). The limit of detection was 5 ng/ml and the lower limit of quantitation was 15 ng/ml. The assay was linear (r=0.9936) over a range of concentrations (5 ng/ml to 5 μg/ml). The assay proved suitable for quantitation of canrenone in a group of patients who had received a 400 mg dose, in whom concentrations in plasma were found to be in the range 5–45 μmol/l.