Related Experiment Video
Updated: Jul 15, 2026
Multi-step Preparation Technique to Recover Multiple Metabolite Compound Classes for In-depth and Informative Metabolomic Analysis
Published on: July 11, 2014
Determination of methadone and its metabolites by high performance liquid chromatography following solid-phase
T L Pierce1, A G Murray, W Hope
1Department of Pharmacology, Victorian College of Pharmacy, Monash University, Parkville, Australia.
Abstract:
This paper describes a rapid, quantitative liquid chromatographic analysis and extraction of methadone and its two major metabolites from rat plasma, using difenoxin as the internal standard. Using a C18 column, resolution of all sample components and the internal standard is achieved with a mobile phase of 25:75 acetonitrile-0.08% diethylamine in 1000 mL water, pH 2.3, at a flow rate of 1.5 mL/min. The injection volume is 100 microL. Standards are linear over the range 25-100 ng, with a lower limit of detection for methadone of 0.25 ng. Within- and between-run coefficients of variation (CV) are 1.24% and 2.94%, respectively. Extraction of methadone and its metabolites from rat plasma uses a solid-phase extraction technique that is highly efficient. Extraction efficiencies of 90.3%, 99.6%, 85.9% and 93.8% were achieved for methadone, its primary and secondary metabolites, and difenoxin, respectively.
