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.