Exploiting sequential injection analysis technique to automate on-line sample treatment and quantitative determination of morphine in human urine

A simple uni-stream sequential injection analysis (SIA) manifold was developed to automate a method for the assay of morphine in human sample. The proposed SIA method includes on-line sample treatment, coupling reaction and spectrophotometric measurement. A rapid algorithm controlled the adopted procedure was critically programmed. For sample treatment, solid-phase extraction (SPE) was carried out into a homemade microcolumn, installed in the SIA manifold. Sufficient sample clean-up, extraction and preconcentration were obtained by SPE. A coupling reaction of morphine with diazonium salt of aniline hydrochloride was adapted to SIA. The product of the reaction, an azo-morphine derivative, was spectrophotometrically detected at 390 nm. Parameters that influenced the efficiency of the proposed method, including solution volumes, diazonium concentration, flow rate and residence time, were optimized. The proposed method was linear in a range of 0.10–2.5 μg ml−1. The limits of detection and quantification were 0.023 and 0.076 μg ml−1, respectively. The detectability of the method was enhanced by the preconcentration and the use of an extended pathlength (50 mm) of a flow cell. The method was validated by an HPLC method. Comparable results with respect to accuracy (recovery 96.3–97.1), repeatability (R.S.D. < 2.4%) and intermediate precision (R.S.D. < 3.1) were gained. The full-automation and miniaturization of the utilized technique offer rapidity, safety in handling urine sample and reagents as well as reduction of reagent and sample volumes. The method is suitable for the application in forensic cases as an initial test and clinical analysis to prevent overdose-induced toxicity.