METHOD DEVELOPMENT AND VALIDATION OF LERCANIDIPINE IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY TANDEM-MASS SPECTROMETRY

Abstract

Objective: To obtain an optimum and validated method for analyzing lercanidipine in plasma using Ultra Performance Liquid Chromatography of Tandem Mass Spectrometry (UPLC-MS/MS).

Methods: The separation was carried out using 1.7μm (2.1 x 100 mm) Waters Acquity^TM UPLC C18 column, a mobile phase of the 0.1% formic acid-methanol mixture (20:80 v/v) with isocratic elution, 30 °C column temperature, 0.2 ml/min flow rate and amlodipine as an internal standard. Mass detection was performed with a positive XBL TQD type Electrospray Ionization (ESI) in Multiple Reaction Monitoring modes. Lercanidipine was detected at m/z value of 612.11>280.27 and amlodipine was detected at m/z value 409.1>238.15. The optimum sample preparation method was a liquid-liquid extraction using 5 ml of n-hexane-ethyl acetate (50:50 v/v), vortex mixed for 3 min, centrifuged at 4000 rpm for 20 min, evaporated with nitrogen at 50 °C for 30 min, and the residue was reconstituted with 100 μl of mobile phase.

Results: The method was linear in the range of 0.025-10 ng/ml with r ≥ 0.9986. Accuracy and precision within-run and between-run met the requirements with %diff and %CV, not exceeding ± 15% and not more than ± 20% for Lower Limit of Quantification (LLOQ) concentration.

Conclusion: It was concluded that the developed method met the requirements of selectivity, carry over, stability, the integrity of dilution, and matrix effects under the Guideline on Bioanalytical Method Validation by the European Medicines Agency in 2011.