Development and validation of a fast and sensitive UHPLC‐ESI‐MS method for the simultaneous quantification of spironolactone and its metabolites in ocular tissues

Abstract

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-H 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.

Ocular Biodistribution of Spironolactone after a Single Intravitreal Injection of a Biodegradable Sustained-Release Polymer in Rats

“…MeT was used as an internal standard (IS). The complete details describing the analytical method development have been published elsewhere …”

“…All calibration curves showed linearity from 10 to 1000 ng/mL ( R > 0.99). This method was previously validated in porcine corneal matrix; limits of detection and limits of quantification (LOQ) were, respectively, 1.6 and 4.9 ng/mL for SPL, 1.0 and 2.9 ng/mL for TMSPL, 2.9 and 8.8 ng/mL for CAN …”

“…The aim of the present study was to investigate, for the first time, the use of hex-PLA polymer as an intravitreal sustained-release delivery system. SPL release from the polymer was quantified over a period of 2 months in vitro and during 1 month in vivo using a novel ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-ESI-MS) analytical method . The changing ocular distribution of SPL and its metabolites [CAN and 7α-thiomethylspironolactone (TMSPL)] in vivo and tolerability were also determined in rats over 1 month following a single IVT inj of 5% SPL–hexPLA.…”

“…The complete details describing the analytical method development have been published elsewhere. 37 Briefly, the UHPLC system consisted of a Waters Acquity UPLC system (Baden-Daẗtwil, Switzerland) including a binary solvent manager, a sample manager with an injection loop volume of 10 μL, and a column manager. Injection volume was set at 5 μL.…”

“…This method was previously validated in porcine corneal matrix; limits of detection and limits of quantification (LOQ) were, respectively, 1.6 and 4.9 ng/mL for SPL, 1.0 and 2.9 ng/mL for TMSPL, 2.9 and 8.8 ng/mL for CAN. 37 Development and Characterization of SPL−hexPLA Formulations. Given the liquid state of the polymer, SPL− hexPLA formulations (3 g) were easily prepared by direct mixing of SPL with the polymer at different drug loadings (1, 2, 3, and 5%, w/w).…”