HPLC-MS/MS method development for the quantitative determination of nifedipine for Caco-2 permeability assay

Aim. Poorly water-soluble drugs such as nifedipineoffer challenging problems in drug formulation as poor solubility is generally associated with poor dissolution characteristics and thus with poor oral bioavailability (BCS class II drugs). Methods of quantitative determination of nifedipine by methods of spectrophotometry and chromatography are described in the scientific literature. However, methods are not developedfor examination of nifedipine from Caco-2 cell monolayers. Caco-2 cell monolayers have been extensively used for years as a tool to test permeability, assess the oral absorption potential and study the absorption mechanism of compounds. Therefore, the aim of this study was to develop and validate an efficient HPLC MS/MS method for determination of nifedipine from Caco-2 cell monolayers.

Materials and methods. Chromatography was achieved on DiscoveryC18, 50 × 2.1 mm, 5 μm column. Samples were chromatographed in a gradient mode (eluent A (acetonitrile – water – formic acid, 5 : 95 : 0.1 v/v), eluent B (acetonitrile – formic acid, 100 : 0.1 v/v)). The initial content of the eluent B is 0%, which increases linearly by 1.0 min to 100% and to 1.01 min returns to the initial 0%. The mobile phase was delivered at a flow rate of 0.4 mL/min into the mass spectrometer ESI chamber. The sample volume was 5 μl.

Results. Under these conditions, nifedipine was eluted at 1.83 min. A linear response function was established at 1 – 100 ng/mL. The regression equation for the analysis was Y = 0.0323x-0.00121 with coefficient of correction (R²) = 0.9987. According to the Caco-2 test results, nifedipine showed high permeability. The within-run coefficients of variation ranged between 0.331% and 0.619% for nifedipine. The within-run percentages of nominal concentrations ranged between 98.80% and 100.63% for nifedipine. The between-run coefficients of variation ranged between 0.332% and 0.615% for nifedipine. The between-run percentages of nominal concentrations ranged between 98.98% and 101.71% for nifedipine. The assay values on both the occasions (intra- and inter-day) were found to be within the accepted limits.

Conclusion. From results of analysis, it can be concluded that developed method is simple and rapid for determination of nifedipine from confluent Caco-2 monolayers and from aqueous solution. Acquired results demonstrate that proposed strategy can be effortlessly and advantageously applied for examination of nifedipine from Caco-2 cell monolayers.