Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography–electrospray ionization–tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)–electrospray ionization–tandem mass spectrometry (ESI–MS–MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 μl samples of rat serum. Deuterated (d3) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins; acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were—morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%).