Simultaneous determination of methadone, buprenorphine and norbuprenorphine in biological fluids for therapeutic drug monitoring purposes

Methadone and buprenorphine are two of the drugs most frequently used for abstinence from illicit opioids and in the treatment of pain. A sensitive and selective high-performance liquid chromatographic method with diode array detection for the simultaneous determination of methadone, buprenorphine and norbuprenorphine has been developed. Separation of the three analytes was obtained by using a reversed-phase column (C8, 250×4.6 mm I.D., 5 μm) and a mobile phase composed of 40% phosphate buffer containing triethylamine, 50% methanol and 10% acetonitrile (final apparent pH = 6.0). Loxapine was used as the internal standard. An accurate pre-treatment procedure of biological samples was developed, using solid-phase extraction with C8 cartridges (100 mg, 1 mL) and needing small amounts of plasma or urine (300 µL). The calibration curves were linear over a working range of 10.0-1500.0 ng/mL for methadone and of 5.0-500.0 ng/mL for buprenorphine and norbuprenorphine in both matrices. The limit of quantitation (LOQ) and the limit of detection (LOD) were 1.0 and 0.4 ng/mL for methadone and 0.5 and 0.2 ng/mL for both buprenorphine and norbuprenorphine, respectively. The method was successfully applied to the analysis of plasma and urine samples from patients undergoing treatment with these drugs. Precision and accuracy results were satisfactory and no interference from endogenous or exogenous compounds was found. The method is suitable for the simultaneous determination of methadone and buprenorphine in human plasma and urine for therapeutic drug monitoring purposes.