Validation of a high-pressure liquid chromatography and fluorescence polarization immunoassay for the determination of methadone in canine plasma.

Methadone is a synthetic opiate derivative that possesses analgesic activity. A modified fluorescence polarization immunoassay (FPIA) method and a high-pressure liquid chromatography (HPLC) method with UV detection were compared for measurement of concentrations of methadone in canine plasma following intravenous and oral methadone administration. The mean+/-SD for accuracy (deviation from actual concentration) and precision (coefficient of variation) when methadone-fortified canine plasma was evaluated with the FPIA method were 3.9+/-3.2% and 4.4+/-2.9%, respectively. The accuracy and precision of the HPLC method were 6.2+/-5.2% and 7.7+/-3.9%, respectively. The limit of quantification for the FPIA and HPLC methods were 25 and 20 ng/mL, respectively. The coefficient of determination (r) between FPIA and HPLC analysis was 0.94 when plasma from dogs dosed with methadone was evaluated. FPIA provides a rapid, sensitive, and specific measurement of methadone in canine plasma following oral and intravenous administration.