Determination of amlodipine in human plasma by electrospray ionization LC-MS/MS method: validation and its stability studies
Anusak Sirikatitham, Kamon Panrat, and Niwan Tanmanee
pp. 455 - 462
Abstract
A sensitive and specific high-performance liquid chromatography combined with electrospray ionization (ESI) tandem mass spectrometry (LC-MS/MS) method, operating in the positive ionization mode, for quantifying of amlodipine in human plasma using tizanidine as internal standard (I.S.) was developed and validated. The analyte and I.S. were extracted by simple one step liquid/liquid extraction with a mixture of diethylether/dichloromethane (70/30, v/v). The chromatographic separation was performed on a C18 analytical column under isocratic conditions using a mixture of 10mM ammonium formate/methanol/acetonitrile (30/50/20, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. Total chromatographic run time was 5.0 min. Detection was performed on a API 2000 QTRAP quadrupole linear ion trap mass spectrometer via turbo ion spray ionization. Quantitation was performed using multiple reaction monitoring (MRM) mode to study parent →
product ion transitions of m/z 409.4 → 238.1 for amlodipine and m/z 254.2 → 44.1 for I.S., respectively. The validation and stability studies were performed according to the Thai FDA guidance for assessment of bioequivalence study in Thailand. The results were within the accepted criteria as stated in the aforementioned guidance. Linearity in plasma was obtained over the concentration range 0.3-15.0 ng/mL, with a coefficient of determination (r2) of 0.9993. Lower limit of quantification (LLOQ) was identifiable and reproducible at 0.3 ng/mL. The within- and between-run precision values were below 10% and the accuracy was ranged from 94.87 to 102.44% at all three quality controls samples levels. The analyte was found to be stable in plasma samples under three freeze-thaw cycles, long-term storage (3 months at -20°C), short-term storage (4 hours at room temperature), post-preparative and stock-solution stability. The robust and rapid LC-MS/MS method has been successfully applied for routine assay to support bioequivalence or pharmacokinetics studies of amlodipine administered as a single oral dose (10 mg tablet) to Thai healthy volunteers.
