A simple UHPLC-PDA method with a fast dilute-and-shot sample preparation for the quantification of canrenone and its prodrug spironolactone in human urine samples.

Introduction: Nowadays, the treatment of hypertension represents an important issue, particularly in developed countries. While in most cases the standard therapeutic approaches, consisting in the administration of 1 to 3 drugs, are adequate to reach adequate blood pressure levels, in some cases more drugs are needed: this condition is called "resistant hypertension". In this context, the administration of a diuretic, such as spironolactone or canrenoate salts, represents a standard practice. Since a reliable discrimination of real cases of resistant hypertension from cases of poor therapeutic adherence is currently difficult to obtain, the adoption of therapeutic drug monitoring has been suggested as a useful tool for this purpose. In this work, the authors developed and validated a simple, cheap and fast dilute-and-shot method with UHPLC-PDA analysis for the quantification of spironolactone and its metabolite canrenone in human urine samples.

Methods: Standards and quality controls were prepared in urine. Only 100 μL of sample were added with 80 μL of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) working solution and 820 μL of phosphate buffer 10 mM pH 3.2 (phase A):acetonitrile (phase B) 90:10 v:v solution. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 μm 2.1 × 150 mm column, with a binary gradient for 11 min at 40 °C.

Results: Accuracy, intra-day and inter-day precision, selectivity and sensitivity fitted FDA guidelines for all analytes (LLOQ and LOD were 156.25 ng/mL and 78.12 ng/mL, respectively, for both analytes) and recovery resulted high and reproducible. Method performances were tested on urine samples from hypertensive patients with good results.

Discussion: This simple analytical method could represent a useful tool for the management of antihypertensive therapy.