An analytical method for cyclosporine using liquid chromatography–mass spectrometry

A liquid chromatographic mass spectrometric (LC‐MS) assay has been developed for cyclosporine A (CyA) in rat plasma using amiodarone as internal standard (IS). Rat plasma (100 µL) containing drug and IS were extracted using liquid–liquid extraction with 4 mL of 95:5 ether:methanol. After evaporation of the organic layer the residue was reconstituted with 500 µL of water. Then the aqueous layer was transferred to LC‐MS sample vials. A 10 µL volume was injected. The analysis was performed on a C 8 column 3.5 µm (2.1 × 50 mm) heated to 60°C with a mobile phase consisting of acetonitrile:methanol:0.2% NH 4 OH (60:20:20) at an isocratic flow‐rate of 0.2 mL/min. The ions used for quantitation of CyA and IS were m / z 1202.8 and 645.9, with retention times of 3.35 and 4.72 min, respectively. Linear relationships ( r 2  > 0.99) were achieved between plasma or blood concentration and peak height ratios (drug:IS) over the concentration range 50–5000 ng/mL. The CV% and mean error were <19%. Based on validation data, the lower limit of quantification for the assay was 50 ng/mL. The reported assay method displayed high measures of linearity, sensitivity, reliability and precision, allowing its applicability in pharmacokinetic studies in rat. Copyright © 2009 John Wiley & Sons, Ltd.