2.6. Determination of plasma nifedipine and dehydronifedipine concentrations

Plasma concentrations of nifedipine and dehydronifedipine were determined using a LC-MS/MS system (Agilent Technologies 1200 series high performance liquid chromatography (Boeblingen, Germany) equipped with a Biosystems-Sciex API 3000 series triple-quadrupole mass spectrometer (Foster City, CA, USA) and an electronspray ionization (ESI) interface). Chromatographic separation was carried out on a C18 column (Waters Symmetry C18, 4.6 × 100 mm, 3.5 μm) using a mobile phase composed of 20% A (2 mM ammonium formate and 0.1% formic acid in water) and 80% B (2 mM ammonium formate and 0.1% formic acid in acetonitrile) at a flow rate of 0.35 ml/min. The column and autosampler were conditioned at 40 °C and 10 °C, respectively. Acquisition for mass spectrometry was performed in positive electrospray ionization mode and the transitions for precursors to the fragmentation ion product are listed in Table 1. The ion-source temperature, the ion spray voltage, and the dwell time were set at 450 °C, 5.5 kV, and 200 ms per channel, respectively. Data were processed using Analyst 1.4.2 software (Applied Biosystems-Sciex, Foster City, CA, USA). Control plasma spiked with nifedipine and dehydronifedipine at the concentrations from 0.005 to 5 μg/ml were analyzed to establish the standard curve for quantification. Assay validation including specificity, selectivity, linearity, stability, recovery and matrix effects of both analytes were conducted according to the Food and Drug Administration (FDA) Guidance for Bioanalytical Method Validation (2001). The lower limit of quantification (LLOQ) was 3.0 ng/ml in plasma.

Multiple reaction monitoring transitions and fragmentation parameters for nifedipine, dehydronifedipine and diazepam.

Diazepam was used as the internal standard for the chromatographic analysis. Chromatographic separation was carried out as described in the methods. DP: declustering potential; FP: focusing potential; CEP: cell entrance potential; CE: collision energy; CXP: cell exit potential; RT: retention time.