Development and validation of a fast and sensitive UHPLC-ESI-MS method for the simultaneous quantification of...

Glucocorticoids are a mainstay for the treatment of immune-mediated conditions and inflammatory diseases. However, their chronic use causes numerous side-effects including delays in corneal and cutaneous wound healing. This is attributed to off-target agonism of the mineralocorticoid receptor, which can be reduced by co-administration of a mineralocorticoid receptor antagonist such as spironolactone. The aim of this study was to develop a fast, selective and sensitive UHPLC-ESI-MS method for the simultaneous quantification of spironolactone, its active metabolites (7α-thiomethylspironolactone and canrenone), the latter's water-soluble prodrug potassium canrenoate and the synthetic glucocorticoid, dexamethasone, in corneal samples (17α-methyltestosterone served as an internal standard). A one-step extraction procedure using MeOH-H₂ O (1:1) was validated and employed to recover the analytes from the corneal tissue. Extracts were centrifuged and the supernatant analyzed under isocratic conditions. Compounds were detected using selected ion recording mode. The method satisfied US Food and Drug Administration guidelines with respect to selectivity, precision and accuracy and displayed linearity from 5 to 1000 ng/mL for all of the analytes. The lower limit of quantitation of the method was 5 ng/mL, making it sufficiently sensitive for quantification of the analytes in samples from in vivo studies.