Development of liquid chromatography tandem mass spectrometry method for determination of spironolactone in human plasma: application to a bioequivalence study of Daewon Spiracton tablet^® (spironolactone 50 mg)

Abstract

A sensitive LC–MS/MS method was developed and fully validated for the determination of spironolactone in human plasma using spironolactone-d₆ as an internal standard (IS) after one-step liquid–liquid extraction with methyl tert-butyl ether: methylene chloride (MC) = 8:2 (v/v). Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the transitions: m/z 341.2 → 107.2 for spironolactone and m/z 347.1 → 107.2 for IS. Chromatographic separation was performed on a Cadenza CD-C₁₈ column (3.0 × 100 mm i.d. 3 µm) with an isocratic mobile phase, which consisted of 0.1 % formic acid in water: methanol (30: 70, v/v), with a gradient flow rate as follow: 0–3.2 m, 320 µL/min; 3.2–3.5 m, 320–180 µL/min; 3.5–6.7 m, 180 µL/min; 6.7–7.0 m, 180–320 µL/min. The calibration curve was linear (correlation coefficients were >0.99) over the concentration range (0.5–150 ng/mL). The intraday and interday precisions ranged 0.89–6.00 and 1.20–10.511 %, respectively, and its accuracies ranged 96.90–105.08 and 97.99–104.13 %, respectively. The devised method was successfully applied in a bioequivalence study of two formulations of spironolactone, Spiracton tablet^® and Aldactone tablet^® in 50 healthy Korean male volunteers following single oral administration.