Development of HPLC-methodology of determination of amiodarone and desethylamiodarone in human blood plasma

Background. Amiodarone is widely used as an antiarrhythmic drug, but its effectiveness is not entirely predictable, and its side effects are not completely controllable. To determine the effective concentration of amiodarone in the blood, a method for quantitative analysis of the drug in the presence of a biological matrix is needed. Objective. To develop a procedure for quantitative determination of amiodarone (AMI) and desethylamiodarone (DEA) in blood plasma by high-performance liquid chromatography (HPLC). Materials and Methods. The procedure was developed using an Agilent 1260 Infinity chromatograph with diode array detector (Agilent, USA) on a Tsunami C18 Pharm column (250×4.6 mm, 5 μm) with proprietary stationary phase. Agilent Zorbax SB C8 precolumn (9.4×15 mm, 7 μm) was used to protect the column from mechanical contaminants. Validation was performed according to OFS.1.1.0012.15 “Validation of Analytical Methods” for the following parameters: selectivity, matrix effect, linearity, accuracy, precision, stability and limits of detection and quantification. Results. The QuEChERS sample preparation was modified to achieve optimal conditions for the extraction of AMI and DEA from the biological matrix. Mobile phase A was phosphate buffer (pH 3, 7.5 mM), mobile phase B was 100% acetonitrile. The separation was performed in gradient mode. Up to 7.00 minutes – the B phase content was 55%, from 7.00 to 7.15 minutes – an increase in B phase content to 85% in order to elute the more hydrophobic components, from 7.15 to 15.00 minutes – the B phase content did not change, followed by a return to the original 50% at 15.15 min. The total analysis time was 18 minutes. Column thermostat temperature was set to 30 °C, flow rate – 1.2 mL/min; injection volume – 80 µL; selective wavelength was 241 nm. The value of retention factor for AMI was 2.25; for DEA – 1.44. Conclusion. The authors have developed and validated the new HPLC procedure for quantitative identification of AMI and DEA in human blood plasma.