Analytical issues in HPLC/MS/MS simultaneous assay of furosemide, spironolactone and canrenone in human plasma samples

A new sensitive HPLC/MS/MS method for simultaneous determination of furosemide, spironolactone and canrenone in human plasma samples is presented. Electrospray ionization source (ESI) has been used. The tandem MS detection was performed under MRM conditions, in the negative ion mode for furosemide and indapamide (internal standard 1) and in the positive ion mode for spironolactone, canrenone and nitrazepam (internal standard 2). A simple plasma protein precipitation with acetonitrile was used as sample preparation technique. The chromatographic separation was carried out under the reversed phase mechanism, on a 250 mm length column packed with octadecyl modified silicagel and thermostated at 35 °C. The column was operated under isocratic conditions (3:7 aqueous 0.1% formic acid and methanol, v/v) at a flow rate of 0.8 mL/min. Quantitation intervals of 20–1600 ng/mL for furosemide and 2–100 ng/mL for spironolactone and canrenone have been concluded through validation. Precision and accuracy were situated within the accepted thresholds (maximum 15% relative standard deviation and ±15% percentage bias). The most sensitive aspects relating to the analytical method development and validation were highlighted and critically assessed in order to reach an objective opinion about the real performances and inherent applicability of the method in bioanalysis.