Abstract
A sensitive, specific and rapid liquid chromatography - tandem mass spectrometry method was developed and validated for the determination of candesartan in human plasma. Analyte was separated from endogenous components present in plasma by solid phase extraction. Chromatographic separation was performed on Gemini C18 analytical column using mobile phase acetonitrile - 5 mM ammonium formate pH 2 (90:10, v/v) at flow rate of 0.3 mL/min. For detection, tandem mass spectrometry in SRM mode with positive electrospray ionization was used. The mass transitions m/z 441.1 > 263.1 and 445.1 > 267.1 were used to determine candesartan by using candesartan-d4 as an internal standard. After development, the method was validated according to the requirements of EMA regulatory guidelines in the concentration range 1 - 400 ng/ml in human plasma. Limit of quantification (LLOQ) was 1 ng/ml. The developed and validated method proved to be very fast and reproducible and was therefore successfully implemented in pharmacokinetic and bioequivalence studies with large number of study samples.
Highlights
Candesartan is an angiotensin II type one receptor (AT1) blocking agent given orally as the prodrug candesartan cilexetil which is rapidly and completely bioactivated by ester hydrolysis to candesartan after gastrointestinal absorption
The presented method was validated for specificity, lower limit of quantification, linearity, precision and accuracy, matrix effect, matrix factor, sample dilution, sample preparation recovery, hemolysis effect, solution stability, stability in plasma and carry-over test performance
All validation procedures were performed according to the acceptance criteria for bioanalytical method validation.[10,11,12]
Summary
Candesartan is an angiotensin II type one receptor (AT1) blocking agent given orally as the prodrug candesartan cilexetil which is rapidly and completely bioactivated by ester hydrolysis to candesartan after gastrointestinal absorption. Several analytical methods were reported for the quantification of candesartan in human plasma.[2,3,4,5,6,7,8,9] Some of these methods were based on high-performance liquid chromatography (HPLC) with UV detection and fluorescence detection.[2,3] Due to the increasing demands in pharmacokinetics studies, analytical methods must be specific, sensitive and rapid These conditions are fully satisfied by LC-MS/MS.[4,5,6,7,8,9] Some reported methods involve the preparation of plasma samples by liquid-liquid extraction[4,5] or solid-phase extraction[3,6,7] or by precipitation of proteins.[8,9] The protein precipitation technique, which is a fast and simple technique, that requires a small sample volume, no preconcentration is obtained and plasma supernatant can contain several matrix components. This article proposes the use of isotopically labeled internal standard candesartan-d4 which has been proven to provide results with excellent precision and accuracy and which can be used with advantage for a reliable and accurate measurement of candesartan in human plasma
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