Automated Fluorometric Method for the Determination of Morphine in Urine

A sensitive and specific automated method for the fluorometric determination of morphine in urine was developed. The analysis is performed at a rate of 40 samples/hr. The method is based on the oxidation of morphine to the fluorescent pseudo-morphine dimer. Morphine is extracted from urine at pH 9.4 with a mixture of organic solvents. The organic phase is washed with a dilute buffer at pH 9.4 and is further extracted with a dilute alkaline solution. Following the third extraction step, 2 equal portions of the alkali phase are buffered to pH 9.4. Potassium ferricyanide solution is added to the sample segment and water is added to the “blank” segment. Each of these solutions enters a separate fluorometer and the fluorescence is recorded. An increment in fluorescence of the sample as compared to the blank indicates the presence of morphine. Concentrations as low as 0.2 μg free morphine/ml urine are easily detected. Five hundred subjects from a Methadone Maintenance Program have been tested. Results show excellent agreement with determinations on the same samples by thin layer chromatography.