Open AccessOptimization and Validation of a Sensitive HPLC–PDA Method for Simultaneous Determination of Torasemide and Spironolactone in Human Plasma using Central Composite Design
Author(s) -
V. Subramanian
Publication year - 2015
Publication title -
acta chimica slovenica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.289
H-Index - 46
eISSN - 1580-3155
pISSN - 1318-0207
DOI - 10.17344/acsi.2014.1262
Subject(s) - chromatography , chemistry , acetonitrile , resolution (logic) , spironolactone , central composite design , high performance liquid chromatography , detection limit , linearity , selectivity , potassium phosphate , potassium , response surface methodology , computer science , aldosterone , biochemistry , physics , organic chemistry , quantum mechanics , artificial intelligence , biology , catalysis , genetics
A sensitive, accurate, precise and rapid HPLC-PDA method was developed and validated for the simultaneous determination of torasemide and spironolactone in human plasma using Design of experiments. Central composite design was used to optimize the method using content of acetonitrile, concentration of buffer and pH of mobile phase as independent variables, while the retention factor of spironolactone, resolution between torasemide and phenobarbitone; and retention time of phenobarbitone were chosen as dependent variables. The chromatographic separation was achieved on Phenomenex C(18) column and the mobile phase comprising 20 mM potassium dihydrogen ortho phosphate buffer (pH-3.2) and acetonitrile in 82.5:17.5 v/v pumped at a flow rate of 1.0 mL min(-1). The method was validated according to USFDA guidelines in terms of selectivity, linearity, accuracy, precision, recovery and stability. The limit of quantitation values were 80 and 50 ng mL(-1) for torasemide and spironolactone respectively. Furthermore, the sensitivity and simplicity of the method suggests the validity of method for routine clinical studies.