Determination of acyclovir in horse plasma and body fluids by high-performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry.

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concen- tration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liq- uid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon® filter was performed. Ganciclovir was used as an internal standard. Analysis was car- ried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL −1 for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5n g mL −1 and at 2 ng mL −1 for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir. Copyright © 2008 John Wiley & Sons, Ltd.