Simultaneous determination of spironolactone and its active metabolite canrenone in human plasma by HPLC‐APCI‐MS

Abstract

A sensitive and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) method for the simultaneous determination of spironolactone and its active metabolite canrenone in human plasma has been developed and validated. After the addition of estazolam as the internal standard (IS), plasma samples were extracted with methylene chloride : ethyl acetate mixture (20 : 80, v/v) and separated by high-performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol-water (57 : 43, v/v). Analytes were determined in a single quadrupole mass spectrometer using an atmospheric pressure chemical ionization (APCI) source. LC-APCI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.25 for spironolactone and canrenone, m/z 295.05 for estazolam. The method was proved to be sensitive and specific by testing six different plasma batches. Calibration curves of spironolactone and canrenone were linear over the range 2-300 ng/ml. The within- and between-batch precisions (relative standard deviation (RSD)%) were lower than 10% and the accuracy ranged from 85 to 115%. The lower limit of quantification (LLOQ) was identifiable and reproducible at 2 ng/ml. The proposed method was successfully applied to study the pharmacokinetics of spironolactone and its major metabolite in healthy male Chinese volunteers.

Determination of Spironolactone and Canrenone in Human Plasma by High-performance Liquid Chromatography with Mass Spectrometry Detection

“…The analytes could be determined with accuracy and precision for quantities starting from 7.8 pg injected into column, in comparison with another method in which a run time of 13 minutes and time and material consuming sample preparation like liquid-liquid extraction were applied in order to determin quantities above 200 pg injected. 3 Another advantage of the actual method is the high sample throughput due to the sample preparation by protein precipitation combined with a short run-time of analysis, in comparison with a recent article in which protein precipitation is used, but large injection volume (corresponding to 133 pg of analytes injected) and long runtime (14 minutes) were necessary to achieve the objectives.…”

“…3,4 However, an important number of papers in which LC-MS determination of SPR from urine (doping agents screening) and milk are described. [5][6][7][8][9][10][11][12] In the present study, we attempted to develop a fast and sensitive LC-MS/MS method able to quantify SPR and CNR in human plasma after oral administration of a single dose of 100 mg (2 × 50 mg) SPR by applying a simple protein precipitation, with a very short run-time.…”

Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples

“…Several HPLC methods have been reported for estimation of CAN in various biomatrices such as serum, plasma and urine [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26], however there are no published methods which have employed whole blood in liquid form or in the form of DBS. Many of the methods have several limitations such as lack of selectivity [10,11], low sensitivity [13,17] or large volume of biomatrix required (1 ml plasma) [18,25] and as such are not suitable for estimation of CAN in very low volume paediatric blood samples.…”

Pharmacologically Active Drug Metabolites: Impact on Drug Discovery and Pharmacotherapy

“…Spironolactone had been shown to generate metabolites that lost the acetyl group but retained the sulfur (Abshagen et al, 1976). A comparison of the concentrations of spironolactone and canrenone (Dong et al, 2006) shows that the metabolite is present at about 3-fold excess at maximum plasma concentration but vastly greater concentrations at later timepoints. When correcting for relative free fractions (Chien et al, 1976) and intrinsic potencies (Funder et al, 1976), it would suggest that canrenone contributes a quarter of the activity of parent.…”