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Abstract
A reversed‐phase high performance liquid chromatographic (RP‐HPLC) method is developed and validated for the determination of methadone, a substitute of heroin used for drug addicts under heroin detoxication treatment in therapeutic communities, as well as its main metabolite EDDP in biological fluids. The analytical column, a Kromasil C18, 5 µm, 250×4 mm, was operated at ambient temperature using isocratic elution with a mixture of CH3CN – 0.025 M CH3COONH4 (90–10 v/v) at a flow rate 1.2 mL/min. Anthracene (1 ng/µL) was used as the internal standard. Inlet pressure was 220 kg/cm2. A DAD detector was monitoring the column eluant at 220 nm.
The limit of detection was 10 ng for EDDP and 20 ng for methadone, per 20 µL injection volume. Linearity held up to 20 ng/µL for methadone and 15 ng/µL for its metabolite. The statistical evaluation of the method was performed in terms of within‐day (n=6) and between‐day (n=8) precision and accuracy, and was found to be satisfactory, with high accuracy and precision results.
The method was successfully applied to biological fluids. Blood serum samples after deproteinization with acetonitrile and solid phase extraction, yielded high recovery rates: 101.5% for EDDP and 103.3% for methadone. Direct analysis of urine provided recovery rates at 97.9% for EDDP and 94.9% for methadone. The developed method can be readily applied to monitoring the levels of methadone and EDDP in biological samples from patients undergoing methadone maintenance therapy, in order to individualize treatment.