Stereoselective determination of methadone in serum by HPLC following solid-phase extraction on disk

A solid-phase extraction (SPE) technique for methadone has been developed using a mixed-mode solid-phase extraction disk which contains both hydrophobic and cation-exchange functional groups. The SPE technique was used to isolate the drug from the biological matrix and to prepare a cleaner sample prior to stereoselective analysis by HPLC on a silica column with covalently bound alpha 1-acid glycoprotein (Chiral-AGP) followed by ultraviolet detection at 205 mm. The within-run precision was less than 5% for the complete method over the therapeutic range. The quantification limit was 25 ng ml-1. The between-run precision was less than 15% at the quantification limit. The between-run precision at other concentrations was less than 8.5% with an accuracy of more than 95%. The mean recovery for R-methadone was 78.5% and the mean recovery for S-methadone was 73.4%. The complete procedure has been validated. This method was successfully used for the analysis of 15 clinical cases.