[Measurement of serum amiodarone by protein precipitation followed by high performance liquid chromatography with...

Our aim was to develop a quantitative method for serum amiodarone measurement using high performance liquid chromatography with ultraviolet photometric detection. We studied previous reports in the literature in order to obtain a simpler method to be used routinely in our TDM unit. Sample preparation was based on protein precipitation adding 2 parts of acetonitrile to 1 part of serum, followed by vortex-agitation for 45 s, incubation at 24 degrees C for 5 min, and centrifugation at 6000 x g for 2 min. Twenty microliters of the supernatant was directly injected into the chromatographic system. A microBondapak CN RP column (3.9 x 150 mm) at 45 degrees C, with a mobile phase consisting of KH2PO4 10 mM/methanol/acetonitrile (40:37:23 v/v/v), pH 3.5, were used. Eluting with a flow rate of 0.6 mL/mm the retention time of amiodarone was approximately 4.9 min. Detection was performed at 242 nm and the quantification was made by peak height comparison with external standards. The mass/response ratio is linear (r2 > 0.99) within a mass range of 2.96 to 18,930 ng of injected amiodarone, which exceeds the requirements for the monitoring of serum levels (0.3 to 6.0 micrograms/mL). Sample storage should be done with acetonitrile-extracted sera at -16 degrees C to avoid degradation. The method is very efficient, linear, sensitive and specific but it also simpler and cheaper than others reported in the literature.