Improved simultaneous quantitation of candesartan and hydrochlorthiazide in human plasma by UPLC-MS/MS and its application in bioequivalence studies

Abstract

A validated ultra-performance liquid chromatography mass spectrometric method (UPLC-MS/MS) was used for the simultaneous quantitation of candesartan (CN) and hydrochlorothiazide (HCT) in human plasma. The analysis was performed on UPLC-MS/MS system using turbo ion spray interface. Negative ions were measured in multiple reaction monitoring (MRM) mode. The analytes were extracted using a liquid-liquid extraction (LLE) method by using 0.1 mL of plasma volume. The lower limit of quantitation for CN and HCT was 1.00 ng/mL whereas the upper limit of quantitation was 499.15 ng/mL and 601.61 ng/mL for CN and HCT respectively. CN d₄ and HCT-¹³Cd₂ were used as the internal standards for CN and HCT respectively. The chromatography was achieved within 2.0 min run time using a C18 Phenomenex, Gemini NX (100 mm×4.6 mm, 5 µm) column with organic mixture:buffer solution (80:20, v/v) at a flow rate of 0.800 mL/min. The method has been successfully applied to establish the bioequivalence of candesartan cilexetil (CNC) and HCT immediate release tablets with reference product in human subjects.

Keywords: Bioequivalence; Candesartan cilexetil; Candesartan cilexetil-hydrochlorothiazide (ATACAND HCT); Hydrochlorothiazide; UPLC–MS/MS.

Fig. 1

The product ion mass spectra of the [M−H]⁻ ions of (A) IS1, (B) CN, (C) IS2 and (D) HCT.

Fig. 2

Representative MRM chromatograms of blank plasma sample (A1 and A2), a plasma sample spiked with CN (B1) and HCT (B2) at the LLOQ level (1.00 ng/mL for both analytes) and a plasma sample from a healthy volunteer 1.5 h after the oral administration of the combination tablet. (C1 for CN and C2 for HCT). CN (left panels A1, B1 and C1) and its ISTD-CN d4 (right panels A1, B1and C1) and HCT (left panels A2, B2and C2) and its ISTD-HCT ¹³Cd₂ (right panels A2, B2 and C2).

Fig. 3

Mean plasma concentration–time curve for CN (A) and HCT (B) compared with standard (Atacand HCT^®).