Method development and validation of amlodipine and cilnidipine in human plasma by using liquid chromatography and tandem mass spectrometry

Abstract

Amlodipine and Cilnidipine in K2EDTA plasma were determined by Liquid Chromatography and Tandem Mass Spectrometry. To improve LC separation and detection sensitivity for the LC-MS technique interface of contiguous stationary phases and LC gradients were enhanced. For separation Acetonitrile was used as eluent. Reversed phase C8 and C18, X-bridge columns were utilized. Telmisartan was used an Internal Standard in this process. For elution Telmisartan needs acidic PH but Amlodipine and Cilnidipine needs neutral PH, dilution with H2O for supernate get afterward extraction. The standards were separated in existence of Telmisartan as internal standard from Amlodipine and Cilnidipine by LC-MS/MS using the MRM transitions. The extracted ion chromatograms were considered from peak ratio of analyte to internal standard. Human plasma without analytes, and solutions containing analytes without human plasma were also analysed. To determine the stability analyte was kept in steady state at room temperature for 24 hrs in plasma. The instrument had operating in positive ion mode. The product ion mass spectra of Amlodipine were 409.1 → 237.7 and Cilnidipine was 493.2 → 117.1. Quadratic regression was used to generate calibration curve by the Analyst 1.5 software program. To obtain the linear response the concentration range is 1-1000ng/mL. For each analyte intra (n=6/batch, 3 days) and inter-batch (n=18) accuracy and precision values were obtained.