Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC–UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC–UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC–MS/MS method. For the HPLC–UV method, good linearity was observed in the range 0–5 μg ml−1, and in the range 0–1 μg ml−1 for the LC–MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml−1 for the HPLC–UV method and the LC–MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml−1 for amiodarone and desethylamiodarone, respectively. The LODs of the LC–MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml−1 for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml−1 for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC–UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC–MS/MS method showed its applicability for single dose pharmacokinetic studies.