Determination of morphine in urine by solid-phase immunoextraction and high-performance liquid chromatography with electrochemical detection

Abstract

The analysis of morphine in biological fluids is of vital interest in monitoring opiate abuse and in drug abuse research. Although methods for analysis of morphine and its metabolites are well established, studies are still being carried out to improve sample preparation procedures as well as detection levels of morphine in biological samples. In this study, morphine-specific immunosorbents were developed to concentrate morphine prior to HPLC analysis. Urine (0.1 ml) was diluted 10-fold with phosphate-buffered saline, pH 7.4 (PBS), loaded onto a solid-phase immunoextraction column and washed with 15 ml PBS followed by elution with 2 ml of elution buffer (40% ethanol in PBS, pH 4). The eluted fraction was analysed for morphine by HPLC–electrochemical detection using a cyanopropyl (CN) analytical column with 25% acetonitrile in phosphate buffer–sodium lauryl sulphate, pH 2.4 as the mobile phase. Duration of the extraction procedure was approximately 40 min. Calibration graphs were linear from 100 ngml−1 to 500 ngml−1 in urine. The inter-assay R.S.D. was <10% and the recovery of morphine from urine was >98%. Immunocolumns demonstrated remarkably high specificity towards morphine showing minimal binding with other opiate metabolites such as codeine, normorphine, norcodeine, morphine-3-glucuronide, morphine-6-glucuronide.