Direct quantification of morphine glucuronides and free morphine in urine by liquid chromatography–tandem mass spectrometry

A simple and robust liquid chromatographic-tandem mass spectrometric method was developed and validated for the direct quantification of the structural isomers morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and morphine in urine. Method development showed that dilution of the urine samples was mandatory to obtain reproducible chromatography and that the use of deuterated internal standards was needed to compensate for ion suppression. Sample preparation consisted of a 20-fold dilution of urine into the internal standard solution. Chromatography was performed on a C₈-column using gradient conditions. The mobile phase consisted of water/methanol, both containing 0.1 % acetic acid and 1 mM ammonium acetate. Calibration curves were constructed between 0.05 and 2 μg/ml. Validation data showed biases ranging from 1.5 to 5.2 % for M3G, −2.7 to −5.9 % for M6G, and 3.7 to 7.1 % for morphine. Imprecision never exceeded 5.2, 5.9, and 5.7 % for M3G, M6G, and morphine, respectively. The applicability of the method was checked by the analysis of 20 urine samples that were analyzed concurrently with a routine gas chromatography-mass spectrometry method. Results showed good correlation between the methods with a correlation coefficient of approximately 0.95.