Bioanalytical methods for the quantification of hydromorphone, fentanyl, norfentanyl, morphine, morphine-3ß-glucuronide and morphine-6ß-glucuronide in human plasma

For the quantitation of hydromorphone, morphine, fentanyl and its metabolite norfentanyl aliquots of 200 μL human potassium EDTA plasma were deproteinized with deuterated internal standards in a mixture of acetonitrile and acetone, followed by a liquid-liquid extraction with 4% ammonium hydroxide and ethyl acetate. Morphine-3ß-glucuronide and morphine-6ß-glucuronide were extracted by a solid phase extraction using 10 mM ammonium carbonate pH 8.8 and a deuterated internal standards solution. Morphine, hydromorphone, fentanyl and norfentanyl were separated on an Aquity UPLC® BEH C18 column 1.7 μm, 100 mm × 2.1 mm at 50 °C. Separation, was achieved on a gradient of methanol with an overall run time of 6 min. The compounds were quantified by triple-quadrupole mass spectrometry in the positive ion electrospray ionization mode. Morphine-3ß-glucuronide and morphine-6ß-glucuronide were separated on a VisionHT C18-P; 3 μm 2.1 × 50 mm, column at 40 °C on a gradient of acetonitrile, with an overall run time of 10 min. Both methods were precise and accurate, with within-run and between-run precisions within acceptable limits and accuracy ranging from 84.0 to 105.5%. The methods were successfully applied to support clinical pharmacological studies in patients treated with opioids for the treatment of moderate to severe cancer-related pain.