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

• Published in: Biomedical chromatography Vol. 23; no. 2; pp. 132 - 140
• Main Authors: Maes, A., Garré, B., Desmet, N., van der Meulen, K., Nauwynck, H., De Backer, P., Croubels, S.
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.02.2009
• Subjects: acyclovir; Acyclovir - administration & dosage; Acyclovir - analogs & derivatives; Acyclovir - analysis; Acyclovir - blood; Administration, Oral; Animals; Antiviral Agents - administration & dosage; Antiviral Agents - analysis; Antiviral Agents - blood; body fluids (nasal fluid; Body Fluids - chemistry; cerebrospinal fluid; Chromatography, High Pressure Liquid - methods; Fluorescence; ganciclovir; Ganciclovir - analysis; Horses - blood; Horses - metabolism; HPLC-fluorescence; Infusions, Intravenous - veterinary; LC-MS/MS; Linear Models; PBMC; plasma; quantification; Reference Standards; Reproducibility of Results; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization - methods; Tandem Mass Spectrometry - methods; validation; Valine - administration & dosage; Valine - analogs & derivatives
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.1093

Two methods are presented for the determination of ‘respectively’ the plasma protein unbound and total concentration 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 liquid 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 carried 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 5 ng 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.